The active center of the protein and its interaction with the ligand. Functioning of proteins
The main property of a protein that ensures its function is selective interaction with a certain substance - a ligand.
Ligands can be substances of different nature, both low molecular weight compounds and macromolecules, including proteins. On protein molecules there are sites to which the ligand is attached - binding centers or active centers. Binding centers are formed from amino acid residues brought together as a result of the formation of secondary and tertiary structures.
The bonds between a protein and a ligand can be non-covalent or covalent. The high specificity of the interaction (“recognition”) of the protein and the ligand is provided by the complementarity of the structure of the binding center with the spatial structure of the ligand.
Complementarity is understood as the chemical and spatial correspondence between the active center of the protein and the ligand. The interaction between protein P and ligand L is described by the equation:
protein + ligand↔ protein-ligand complex.
1. The main physicochemical properties of proteins are molecular weight, electric charge and solubility in water. The molecular weight of proteins can vary considerably. For example, the hormone insulin has a molecular weight of about 6 thousand Da, while immunoglobulin M has a molecular weight of about 1 million Da. The molecular weight of a protein depends on the number of amino acid residues that make up its composition, as well as the mass of non-amino acid components. The mass of one amino acid residue averages 110 Da. Thus, knowing the number of amino acid residues in a protein, one can estimate its molecular weight and vice versa (N.N. Mushkambarov, 1995). The electrical charge of a protein is determined by the ratio of positively and negatively charged groups on the surface of its molecule. The charge of a protein particle depends on the pH of the medium. The concept of "isoelectric point" is used to characterize a protein. Isoelectric point (pI) - the pH value of the medium at which the total charge of the protein particle zero. At the isoelectric point, proteins are the least stable in solution and precipitate out easily. The value of pI depends on the ratio of acidic and basic amino acids in the protein. For proteins and peptides with a predominance of acidic amino acids (negatively charged at pH 7.0), the pI value is in an acidic environment; for proteins and peptides with a predominance of basic amino acids (positively charged at pH 7.0), the pI value is in an acidic environment. The isoelectric point is a characteristic constant of proteins, its value for most animal tissue proteins ranges from 5.5 to 7.0, which indicates the predominance of acidic amino acids in their composition. However, in nature there are proteins whose isoelectric point value lies at the extreme pH values of the medium. In particular, the pI value of pepsin (an enzyme of gastric juice) is 1, and that of lysozyme (an enzyme that breaks down the cell wall of microorganisms) is about 11. The molecular weight and isoelectric point values of some proteins are shown in Table 1.4. Table 1.4 Some constants of blood plasma and tissue proteins
Solubility of proteins in water. From the course of biophysical chemistry, it is known that proteins, as high-molecular compounds, form colloidal solutions. The stability of protein solutions in water is determined by the following factors:
Keep in mind that under the influence of various physical and chemical factors, precipitation of proteins from colloidal solutions can occur. Distinguish:
Note that the following mechanisms may underlie protein precipitation reactions:
Most often, the action of factors causing protein precipitation is characterized by a combination of two or all three of these mechanisms. biological activity. The functioning of any protein is based on its ability to selectively interact with strictly defined molecules or ions - ligands. For example, for enzymes that catalyze chemical reactions, ligands will be substances involved in these reactions (substrates), as well as cofactors, activators, and inhibitors. For transport proteins, the ligands are the transported substances, and so on. The ligand is capable of interacting with a specific site of the protein molecule - the binding center or the active center. This center is formed by spatially close amino acid radicals at the level of the protein tertiary structure. The ability of the ligand to interact with the binding center is due to their complementarity, that is, the mutual complementarity of their spatial structure (similar to the “key-lock” interaction). Non-covalent (hydrogen, ionic, hydrophobic) bonds are formed between the functional groups of the ligand and the binding site. The complementarity of the ligand and the binding site can explain the high specificity (selectivity) of the protein-ligand interaction. So, different proteins differ from each other in their physicochemical properties and biological activity. Methods for separating protein mixtures into fractions and isolating individual enzymatic proteins are based on these differences. These methods are widely used in medical biochemistry and biotechnology. 2. Protein denaturation- this is a change in the native (natural) physicochemical and, most importantly, biological properties of a protein due to a violation of its quaternary, tertiary, and even secondary structure. Protein denaturation can be caused by:
Denatured proteins are characterized by:
Please note that under certain conditions it is possible to restore the original (native) protein conformation after the removal of the factor that caused the denaturation. This process is called regeneration. Remember some examples of the use of the process of protein denaturation in medicine:
|
4(1). Hemoglobin is an allosteric protein. Conformational changes in the hemoglobin molecule. cooperative effect. Hemoglobin affinity regulators for oxygen. Structural and functional differences between myoglobin and hemoglobin.
Hemoglobin: an allosteric protein
The evolutionary transition from monomeric myoglobin to tetrameric hemoglobin was accompanied by the appearance of new properties. The hemoglobin molecule is much more complex than the myoglobin molecule. First of all, hemoglobin, in addition to 0 2, transports H + and CO 2. Secondly, the binding of oxygen by hemoglobin is regulated by specific components of the internal environment, namely H + , CO 2 and organic phosphorus compounds. These regulators have a strong influence on the ability of hemoglobin to bind oxygen, despite the fact that they attach to the protein at sites far from the heme. In general, the so-called allosteric interaction, those. interaction between spatially separated regions occurs in many proteins. Allosteric effects play essential role in the regulation and integration of molecular processes in biological systems. Hemoglobin is the most studied allosteric protein, and therefore it makes sense to consider its structure and function in more detail.
CONFORMATIONAL CHANGES IN HEMOGLOBIN
The binding of oxygen is accompanied by the rupture of salt
bonds formed by terminal carboxyl groups
subunits (Fig. 7) This facilitates the binding of the following molecules
oxygen, since it requires the breaking of a smaller number
salt bonds. These changes have a significant impact on
secondary, tertiary and especially quaternary structure
hemoglobin. In this case, one A / B pair of subunits turns
with respect to another A/B pair, which leads to the compactization
tetramer and increased heme affinity for oxygen (Figs. 8 and 9).
CONFORMATIONAL CHANGES IN THE ENVIRONMENT OF THE HEMOGROUP
Oxygenation of hemoglobin is accompanied by structural
changes in the hemogroup environment. When oxygenated, the atom
iron, which in deoxyhemoglobin protruded 0.06 nm from
plane of the heme ring, draws into this plane (Fig.
10). Following the iron atom, it moves closer to the heme
proximal histidine (F8), as well as related neighboring
The hemoglobin molecule can be in two forms - tense and relaxed. The relaxed form of hemoglobin tends to be saturated with oxygen 70 times faster than the tense one. The change in the fractions of the tense and relaxed forms in the total amount of hemoglobin in the blood determines the S-shaped form of the oxyhemoglobin dissociation curve, and, consequently, the so-called hemoglobin affinity for oxygen. If the probability of a transition from a tense form of hemoglobin to a relaxed one is greater, then the affinity of hemoglobin for oxygen increases, and vice versa. The probability of formation of these hemoglobin fractions changes up or down under the influence of several factors. The main factor is the binding of oxygen to the heme group of the hemoglobin molecule. At the same time, the more hemoglobin hemoglobin groups bind oxygen in erythrocytes, the easier the transition of the hemoglobin molecule to a relaxed form becomes and the higher their affinity for oxygen. Therefore, at low P02, which occurs in metabolically active tissues, the affinity of hemoglobin for oxygen is lower, and at high P02, it is higher. As soon as hemoglobin captures oxygen, its affinity for oxygen increases and the hemoglobin molecule becomes saturated when bound to four oxygen molecules. When erythrocytes containing hemoglobin reach the tissues, oxygen from the erythrocytes diffuses into the cells. In muscles, it enters a kind of oxygen depot - into myoglobin molecules, from which oxygen is used in the biological oxidation of muscles. Diffusion of oxygen from erythrocyte hemoglobin into tissues is due to low P02 in tissues - 35 mm Hg. Art. Inside tissue cells, the oxygen tension required to maintain normal metabolism is even smaller - no more than 1 kPa. Therefore, oxygen by diffusion from capillaries reaches metabolically active cells. Some tissues are adapted to the low content of PO2 in the blood capillaries, which is compensated by the high density of capillaries per unit volume of tissues. For example, in skeletal and cardiac muscle, capillary P02 can drop extremely rapidly during contraction. Muscle cells contain the protein myoglobin, which has a higher affinity for oxygen than hemoglobin. Myoglobin is intensively saturated with oxygen and promotes its diffusion from the blood into the skeletal and cardiac muscles, where it causes the processes of biological oxidation. These tissues are able to extract up to 70% of oxygen from the blood passing through them, which is due to a decrease in the affinity of hemoglobin for oxygen under the influence of tissue temperature and pH. The effect of pH and temperature on the affinity of hemoglobin for oxygen. Hemoglobin molecules are able to react with hydrogen ions, as a result of this reaction, a decrease in the affinity of hemoglobin for oxygen occurs. When hemoglobin saturation is less than 100%, low pH reduces the binding of oxygen to hemoglobin - the oxyhemoglobin dissociation curve shifts to the right along the x-axis. This change in the properties of hemoglobin under the influence of hydrogen ions is called the Bohr effect. Metabolically active tissues produce acids such as lactic acid and CO2. If the pH of the blood plasma decreases from normal 7.4 to 7.2, which occurs during muscle contraction, then the oxygen concentration in it will increase due to the Bohr effect. For example, at a constant pH of 7.4, the blood would give up about 45% oxygen, i.e., the saturation of hemoglobin with oxygen would decrease to 55%. However, when the pH drops to 7.2, the dissociation curve shifts along the x-axis to the right. As a result, oxygen saturation of hemoglobin drops to 40%, i.e., blood can give up to 60% oxygen to tissues, which is 1/3 more than at a constant pH. Metabolically active tissues increase heat production. An increase in tissue temperature during physical work changes the ratio of hemoglobin fractions in erythrocytes and causes a shift in the oxyhemoglobin dissociation curve to the right along the x axis. As a result large quantity oxygen will be released from the hemoglobin of erythrocytes and enter the tissues. The effect of 2,3-diphosphoglycerate (2,3-DPG) on the affinity of hemoglobin for oxygen. Under certain physiological conditions, for example, when P02 in the blood falls below normal (hypoxia) as a result of a person being at a high altitude above sea level, the supply of oxygen to tissues becomes insufficient. During hypoxia, the affinity of hemoglobin for oxygen may decrease due to an increase in the content of 2,3-DPG in erythrocytes. In contrast to the Bohr effect, the decrease in the affinity of hemoglobin for oxygen under the influence of 2,3-DPG is not reversible in the capillaries of the lungs. However, when blood moves through the capillaries of the lungs, the effect of 2,3-DPG on reducing the formation of oxyhemoglobin in erythrocytes (the flat part of the oxyhemoglobin dissociation curve) is less pronounced than the release of oxygen under the influence of 2,3-DPG in tissues (sloping part of the curve), which provides normal oxygen supply to tissues
The native three-dimensional structure is established as a result of the action of a number of energy and entropy factors. A change in the conformational state of a protein molecule due to various external influences (pH, temperature, ionic composition) is also reflected in its functional activity. Conformational rearrangements occur very quickly. At the first stages, they have a local microconformational character, causing displacements of only individual atomic groups. The spread of such local displacements to other regions of the macromolecular structure will already lead to a general conformational change in the biopolymer molecule.
myoglobin- consists of one polypeptide chain, including 153 amino acid residues, and one iron porphyrin group (heme) per molecule. Myoglobin refers to hemoproteins that can reversibly bind oxygen; in skeletal muscle cells, it is responsible for the reservation of oxygen, as well as for increasing the rate of its diffusion through the cells. Phylogenetically, myoglobin is the precursor of hemoglobin. The molecule does not contain disulfide bonds and is characterized by a-helicity by 77%. The heme responsible for oxygen binding is located in a "hydrophobic pocket" formed by special amino acids intended for this purpose. Heme is a protoporphyrin macrocycle with a coordinatingly bound ferrous ion located in the center of the molecule. This spatial fixation of the heme makes it possible to bind an oxygen molecule as the sixth ligand.
Hemoglobin- "respiratory" blood protein. It transports oxygen through the circulatory system of the lungs to other organs and consumption centers. The hemoglobin molecule consists of four pairwise identical polypeptide chains, each of which carries a heme. The polypeptide chains of hemoglobin are called a and b , and the symmetrical structure of the molecule is written as a 2 b 2 . The formation of a quaternary structure is carried out by hydrophobic interactions between individual polypeptide chains. When oxygen is added to heme, oxyhemoglobin is formed, the quaternary structure of which differs only slightly from the non-oxygenated form.
The addition of oxygen induces a number of conformational changes in the Hb molecule. The binding of oxygen with the transfer of the Fe 2+ ion to the low-spin state is accompanied by a simultaneous displacement of iron into the plane of the heme group. There is a gradual rupture of salt bridges between a-subunits. The distance between hemes of a-subunits increases, and between hemes of b-subunits is reduced. In general, oxygenation transforms each of the subunits from deoxy and oxy conformations. The rupture of four salt bridges out of six during oxygenation of the first two a-subunits contributes to the rupture of the other two bridges and, therefore, facilitates the attachment of the following oxygen molecules to the remaining subunits, increasing their affinity for oxygen by several hundred times. This is the cooperative nature of accession.
5(1). Primary and secondary structures of DNA. Chargaff rules. The principle of complementarity. Types of bonds in the DNA molecule. The biological role of DNA. Molecular diseases are the result of gene mutations.
Primary structure of DNA - the order of alternation of deoxyribonucleoside monophosphates (dNMP) in the polynucleotide chain.
Each phosphate group in the polynucleotide chain, with the exception of the phosphorus residue at the 5 "-end of the molecule, participates in the formation of two ester bonds involving 3"- and 5"-carbon atoms of two neighboring deoxyriboses, therefore the bond between monomers is denoted by 3", 5"- phosphodiester.
The terminal nucleotides of DNA are distinguished by structure: at the 5 "end there is a phosphate group, and at the 3" end of the chain there is a free OH group. These ends are called 5" and 3" ends. The linear sequence of deoxyribonucleotides in the DNA polymer chain is usually abbreviated using a one-letter code, for example -A-G-C-T-T-A-C-A- from 5 "- to 3"-end.
Each nucleic acid monomer contains a phosphoric acid residue. At pH 7, the phosphate group is fully ionized, so in vivo Nucleic acids exist as polyanions (have multiple negative charges). Residues of pentoses also exhibit hydrophilic properties. Nitrogenous bases are almost insoluble in water, but some atoms of the purine and pyrimidine rings are able to form hydrogen bonds.
secondary structure DNA. In 1953, J. Watson and F. Crick proposed a model of the spatial structure of DNA. According to this model, the DNA molecule has the shape of a helix formed by two polynucleotide chains twisted relative to each other and around a common axis. double helix right-handed, polynucleotide chain in it antiparallel(Fig. 4-6), i.e. if one of them is oriented in the direction 3"→5", then the second one is oriented in the direction 5"→3". Therefore, at each end
Rice. 4-6. Double helix of DNA. DNA molecules consist of two antiparallel strands with a complementary nucpeotide sequence. The chains are twisted relative to each other in a right-handed helix so that there are approximately 10 base pairs per turn.
DNA molecules are located at the 5" end of one strand and the 3" end of the other strand.
All bases of DNA chains are located inside the double helix, and the pentose phosphate backbone is outside. Polynucleotide chains are held relative to each other due to hydrogen bonds between complementary purine and pyrimidine nitrogenous bases A and T (two bonds) and between G and C (three bonds) (Fig. 4-7). With this combination, each
Rice. 4-7. Purine-pyrimidine base pairs in DNA.
the pair contains three rings, so the total size of these base pairs is the same along the entire length of the molecule. Hydrogen bonds with other combinations of bases in a pair are possible, but they are much weaker. The nucleotide sequence of one chain is completely complementary to the nucleotide sequence of the second chain. Therefore, according to the Chargaff rule (Erwin Chargaff in 1951 established patterns in the ratio of purine and pyrimidine bases in a DNA molecule), the number of purine bases (A + G) is equal to the number of pyrimidine bases (T + C).
Complementary bases are stacked at the core of the helix. Between the bases of a double-stranded molecule in a stack, hydrophobic interactions, stabilizing the double helix.
Such a structure excludes the contact of nitrogenous residues with water, but the base stack cannot be absolutely vertical. The base pairs are slightly offset from each other. In the formed structure, two grooves are distinguished - a large one, 2.2 nm wide, and a small one, 1.2 nm wide. Nitrogenous bases in the region of the major and minor grooves interact with specific proteins involved in the organization of the chromatin structure.
Chargaff rules- a system of empirically identified rules that describe the quantitative relationships between different types of nitrogenous bases in DNA. They were formulated as a result of the work of a group of biochemist Erwin Chargaff in 1949-1951.
Prior to the work of the Chargaff group, the so-called “tetranucleotide” theory dominated, according to which DNA consists of repeating blocks of four different nitrogenous bases (adenine, thymine, guanine and cytosine). Chargaff and co-workers were able to separate DNA nucleotides using paper chromatography and determine the exact quantitative ratios of different types of nucleotides. They differed significantly from the equimolar ones that would be expected if all four bases were represented in equal proportions. The relationships identified by Chargaff for adenine (A), thymine (T), guanine (G) and cytosine (C) were as follows:
1. The amount of adenine is equal to the amount of thymine, and guanine is equal to cytosine: A=T, G=C.
2. The number of purines is equal to the number of pyrimidines: A + G = T + C.
3. The number of bases with amino groups in position 6 is equal to the number of bases with keto groups in position 6: A+C=G+T.
However, the ratio (A + T): (G + C) may be different in DNA different types. In some, AT pairs predominate, in others - HC.
Chargaff's rules, along with X-ray diffraction data, played a decisive role in deciphering the structure of DNA by J. Watson and Francis Crick.
Complementarity(V chemistry, molecular biology And genetics) - mutual correspondence of molecules biopolymers or their fragments, which ensures the formation of bonds between spatially complementary (complementary) fragments of molecules or their structural fragments due to supramolecular interactions(formation of hydrogen bonds, hydrophobic interactions, electrostatic interactions of charged functional groups, etc.).
The interaction of complementary fragments or biopolymers is not accompanied by the formation of a covalent chemical bond between complementary fragments, however, due to the spatial mutual correspondence of complementary fragments, it leads to the formation of many relatively weak bonds (hydrogen and van der Waals) with a sufficiently large total energy, which leads to the formation of stable molecular complexes.
At the same time, it should be noted that the mechanism of the catalytic activity of enzymes is determined by the complementarity of the enzyme and the transition state or intermediate product of the catalyzed reaction - and in this case, reversible formation of a chemical bond can occur.
Nucleic acid complementarity
When nucleic acids- both oligo- and polynucleotide nitrogenous bases nucleotides capable due to education hydrogen bonds form paired complexes adenine-thymine(or uracil V RNA) And guanine-cytosine when chains interact nucleic acids. This interaction plays a key role in a number of fundamental processes storage and transmission of genetic information: DNA replication, which ensures the transfer of genetic information during cell division, transcriptions DNA to RNA during synthesis proteins, encoded by DNA gene, storage of genetic information in double-stranded DNA and DNA repair processes when it is damaged.
The principle of complementarity is used in DNA synthesis. This is a strict correspondence of the compound of nitrogenous bases connected by hydrogen bonds, in which: A-T ( adenine connects with thymin) G-C ( Guanine connects with Cytosine)
Enzymatic catalysis
Complementary enzyme-substrate binding is a key factor in the mechanism of enzymatic activity and, in contrast to the situations described above with the formation of chemically unbound complexes, can lead to the initiation chemical reaction- in case of connection enzyme with the substrate, the complementarity is relatively low, however, with high complementarity to the transition reaction state of the substrate, this state is stabilized, which leads to the effect of the catalytic activity of enzymes: such stabilization of the transition state is equivalent to a decrease in activation energy and, accordingly, a sharp increase in the reaction rate.
Ppt%5C34928-slozhnye_belki_ch1_1.jpg" alt=">The active center of the protein and its interaction with the ligand. In the process of formation of the tertiary structure"> Активный центр белка и его взаимодействие с лигандом. В процессе формирования третичной структуры на поверхности функционально !} active protein, usually in a recess, a site is formed formed by amino acid radicals that are far apart in the primary structure. This site, which has a unique structure for a given protein and is able to specifically interact with a certain molecule or group of similar molecules, is called the protein binding site with a ligand or active site. Ligands are molecules that interact with proteins.
Ppt%5C34928-slozhnye_belki_ch1_2.jpg" alt=">Ligand can be either a low molecular weight or a high molecular weight (macromolecule) substance, including"> Лигандом может быть как низкомолекулярное, так и высокомолекулярное (макромолекула) вещество, в том числе и другой белок. Лигандами являются субстраты ферментов, кофакторы, ингибиторы и активаторы ферментов, протомеры в олигомерном белке и т.д.!}
Ppt%5C34928-slozhnye_belki_ch1_3.jpg" alt=">The high specificity of the interaction of the protein with the ligand is ensured by the complementarity of the structure of the active center with the structure of the ligand.">!}
Ppt%5C34928-slozhnye_belki_ch1_4.jpg" alt=">Complementarity is the spatial and chemical correspondence of interacting surfaces. The active center should not only"> Комплементарность - это пространственное и химическое соответствие взаимодействующих поверхностей. Активный центр должен не только пространственно соответствовать входящему в него лиганду, но и между функциональными группами радикалов, входящих в активный центр, и лигандом должны образоваться связи чаще всего нековалентные (ионные, водородные, а также гидрофобные взаимодействия), которые удерживают лиганд в активном центре.!}
Ppt%5C34928-slozhnye_belki_ch1_5.jpg" alt=">Complementary protein–ligand interaction">!}
Ppt%5C34928-slozhnye_belki_ch1_6.jpg" alt=">">
Ppt%5C34928-slozhnye_belki_ch1_7.jpg" alt=">">
Ppt%5C34928-slozhnye_belki_ch1_8.jpg" alt=">PROTEIN CLASSIFICATION 1. Simple proteins consist only of amino acids. 2. Complex proteins (holoproteins)"> КЛАССИФИКАЦИЯ БЕЛКОВ 1. Простые белки состоят только из аминокислот. 2. Сложные белки (холопротеины) содержат белковую часть (апопротеин) и небелковую (простетическую) группу.!}
Ppt%5C34928-slozhnye_belki_ch1_9.jpg" alt=">Various organic (lipids, carbohydrates) and inorganic (metals) substances can act as a prosthetic group."> В качестве простетической группы могут выступать различные органические (липиды, углеводы) и неорганические (металлы) вещества. Связь между простетической группой и апопротеином может быть как ковалентная, так и нековалентная. Простетическую группу порой можно рассматривать в качестве лиганда. Наличие небелковой части обеспечивает выполнение белком его функции. При утрате простетической группы холопротеин теряет свою активность.!}
Ppt%5C34928-slozhnye_belki_ch1_10.jpg" alt=">Complex proteins - chromoproteins - nucleoproteins - lipoproteins - phosphoproteins - glycoproteins - metalloproteins">!}
Ppt%5C34928-slozhnye_belki_ch1_11.jpg" alt=">Metalloproteins include holoenzymes containing non-heme coordinated metal ions. Metalloproteins include proteins,"> Металлопротеинам можно отнести холоферменты, содержащие негемовые координационно связанные ионы металлов. Среди металлопротеинов есть белки, выполняющие депонирующие и транспортные функции (например, железосодержащие ферритин и трансферрин) и ферменты (например, цинксодержащая карбоангидраза и различные супероксиддисмутазы, содержащие в качестве активных центров ионы меди, марганца, железа и других металлов). Но и хромопротеины, содержащие ионы металлов, также можно отнести к металлопротеинам.!}
Ppt%5C34928-slozhnye_belki_ch1_12.jpg" alt=">Metalloproteins are often enzymes. Metal ions in this case: - participate in substrate orientation"> Металлопротеины часто являются ферментами. Ионы металлов в этом случае: - участвуют в ориентации субстрата в активном центре фермента, входят в состав активного центра фермента и участвуют в катализе, являясь, например, акцепторами электронов на определенной стадии ферментативной реакции. Часто ион металла в составе фермента называют кофактором.!}
Ppt%5C34928-slozhnye_belki_ch1_13.jpg" alt=">Enzymatic metalloproteins include proteins containing, for example: - copper - cytochrome oxidase, in the complex"> К ферментативным металлопротеинам относятся белки, содержащие например: - медь – цитохромоксидаза, в комплексе с другими ферментами дыхательной цепи митохондрий участвует в синтезе АТФ, - железо – ферритин, депонирующий железо в клетке, трансферрин, переносящий железо в крови, каталаза, обезвреживающая перекись водорода, - цинк – алкогольдегидрогеназа, обеспечивающая метаболизм этанола и других спиртов, лактатдегидрогеназа, участвующая в метаболизме молочной кислоты, - карбоангидраза, образующая угольную кислоту из CO2 и H2O, - щелочная фосфатаза, гидролизующая фосфорные эфиры различных соединений, - α2-макроглобулин, антипротеазный белок крови. - селен – тиреопероксидаза, участвующая в синтезе гормонов щитовидной железы, антиоксидантный фермент глутатионпероксидаза, - кальций – α-амилаза слюны и панкреатического сока, гидролизующая крахмал.!}
Ppt%5C34928-slozhnye_belki_ch1_14.jpg" alt=">Ferritin">!}
Ppt%5C34928-slozhnye_belki_ch1_15.jpg" alt=">Phosphoproteins are proteins that have a phosphate group. It binds to the peptide chain"> Фосфопротеины – это белки, в которых присутствует фосфатная группа. Она связывается с пептидной цепью через остатки тирозина, серина и треонина, т.е. тех аминокислот, которые содержат ОН-группу. Способ присоединения фосфата к белку на примере серина и тирозина!}
Ppt%5C34928-slozhnye_belki_ch1_16.jpg" alt=">Phosphoric acid can perform: - Structural role, giving a charge, solubility and changing properties"> Фосфорная кислота может выполнять: - Структурную роль, придавая заряд, растворимость и изменяя свойства белка, например, в казеине молока, яичном альбумине. Наличие остатков фосфорной кислоты способствует связыванию кальция, что необходимо для формирования, например, костной ткани. - Функциональную роль. В клетке присутствует много белков, которые связаны с фосфатом не постоянно, а в зависимости от активности метаболизма. Белок может многократно переходить в фосфорилированную или в дефосфорилированную форму, что играет регулирующую роль в его работе.!}
Ppt%5C34928-slozhnye_belki_ch1_17.jpg" alt=">Phosphorylation is the process of transferring a phosphoric acid residue from a donor phosphorylating agent to a substrate, usually"> Фосфорилирование - процесс переноса остатка фосфорной кислоты от фосфорилирующего агента-донора к субстрату, как правило, катализируемый ферментами (киназами) и ведущий к образованию эфиров фосфорной кислоты. Дефосфорилирование (утрату остатка фосфорной кислоты) катализируют фосфатазы. АТФ + R-OH → АДФ + R-OPO3H2 R-OPO3H2 + Н2О → R-OH + Н3РО4!}
Ppt%5C34928-slozhnye_belki_ch1_18.jpg" alt="> Examples: 1) enzymes glycogen synthase and glycogen phosphorylase 2) histones in the phosphorylated state bind less strongly"> Примеры: 1) ферменты гликогенсинтаза и гликогенфосфорилаза 2) гистоны в фосфорилированном состоянии менее прочно связываются с ДНК и активность генома возрастает. Изменение конформации белка в фосфорилированном и дефосфорилированном состоянии!}
Ppt%5C34928-slozhnye_belki_ch1_19.jpg" alt=">Lipoproteins contain non-covalently bound lipids as a prosthetic part. Lipids, in particular"> Липопротеины содержат в качестве простетической части нековалентно связанные липиды. Липиды, в частности жиры, холестерол и его эфиры не растворяются в водных фазах организма, поэтому транспорт их кровью и лимфой осуществляется в виде комплексов с белками и фосфолипидами, которые называются липопротеинами.!}
Ppt%5C34928-slozhnye_belki_ch1_20.jpg" alt=">All lipoproteins have a similar structure: the core consists of hydrophobic molecules: triacylglycerols, cholesterol esters, and"> Все липопротеины имеют сходное строение: ядро состоит из гидрофобных молекул: триацилглицеролов, эфиров холестерола, а на поверхности находится монослой фосфолипидов, полярные группы которых обращены к воде, а гидрофобные погружены в гидрофобное ядро липопротеина. Кроме фосфолипидов, на поверхности находятся белки – аполипопротеины (апобелками). Их выделяют несколько видов: А, В, С, D. В каждом типе липопротеинов преобладают соответствующие ему апобелки. Аполипопротеины выполняют различные функции. Интегральные аполипопротеины являются структурными компонентами. Периферические аполипопротеины в плазме крови могут передаваться от одного типа липопротеинов к другим, определяя их дальнейшие превращения.!}
Ppt%5C34928-slozhnye_belki_ch1_21.jpg" alt=">Lipoprotein structure scheme Lipoprotein structure">!}
Ppt%5C34928-slozhnye_belki_ch1_22.jpg" alt=">The structure of blood plasma lipoproteins">!}
Ppt%5C34928-slozhnye_belki_ch1_23.jpg" alt="> There are four main classes of lipoproteins: high density lipoproteins (HDL), low density lipoproteins (LDL),"> Выделяют четыре основных класса липопротеинов: -липопротеины высокой плотности (ЛПВП), -липопротеины низкой плотности (ЛПНП), -липопротеины очень низкой плотности (ЛПОНП), -хиломикроны (ХМ). Каждый из типов ЛП образуется в разных тканях и транспортирует определённые липиды. Концентрация и соотношение в крови тех или иных липопротеинов играют ведущую роль в возникновении такой распространенной сосудистой патологии как атеросклероз. ЛПВП являются антиатерогенными, ЛПНП и ЛПОНП – атерогенными.!}
Ppt%5C34928-slozhnye_belki_ch1_24.jpg" alt=">">
Ppt%5C34928-slozhnye_belki_ch1_25.jpg" alt=">Glycoproteins or glycoconjugates are proteins containing a carbohydrate component covalently attached to a polypeptide backbone."> Гликопротеины или, гликоконъюгаты – это белки, содержащие углеводный компонент, ковалентно присоединенный к полипептидной основе. Содержание углеводов в них варьирует от 1% до 98% по массе. Два подкласса белков, содержащих углеводы: ■ протеогликаны ■ гликопротеины!}
Description="">
Ppt%5C34928-slozhnye_belki_ch1_27.jpg" alt="> Glycoproteins are characterized by a low content of carbohydrates, which are attached: - by an N-glycosidic bond to the NH2 group of some"> Для гликопротеинов характерно невысокое содержание углеводов, которые присоединены: - N-гликозидной связью к NН2-группе какого-нибудь аминокислотного остатка, например, аспарагина; - О-гликозидной связью к гидроксильной группе остатка серина, треонина,тирозина, гидроксилизина.!}
Ppt%5C34928-slozhnye_belki_ch1_28.jpg" alt=">Formation of O- and N-glycosidic bonds in glycoproteins. 1 - N-glycosidic bond between the amide group"> Образование О- и N-гликозидных связей в гликопротеинах. 1 - N-гликозидная связь между амидной группой аспарагина и ОН-группой моносахарида; 2 - О-гликозидная связь между ОН-группой серина и ОН-группой моносахарида.!}
Ppt%5C34928-slozhnye_belki_ch1_29.jpg" alt=">A way to attach a carbohydrate to a protein">!}
Ppt%5C34928-slozhnye_belki_ch1_30.jpg" alt=">The functions of glycoproteins are: 1. Structural - bacterial cell wall, bone matrix, for example, collagen, elastin."> Функцией гликопротеинов являются: 1. Структурная – клеточная стенка бактерий, костный матрикс, например, коллаген, эластин. 2. Защитная – например, антитела, интерферон, факторы свертывания крови (протромбин, фибриноген). 3. Рецепторная – присоединение эффектора приводит к изменению конформации белка-рецептора, что вызывает внутриклеточный ответ. 4. Гормональная – гонадотропный, адренокортикотропный и тиреотропный гормоны. 5. Ферментативная – холинэстераза, нуклеаза. 6. Транспортная – перенос веществ в крови и через мембраны, например, трансферрин, транскортин, альбумин, Na+,К+-АТФаза.!}
Ppt%5C34928-slozhnye_belki_ch1_31.jpg" alt=">Scheme of the receptor protein structure">!}
Ppt%5C34928-slozhnye_belki_ch1_32.jpg" alt=">Chromoproteins are a collective name for complex proteins with colored prosthetic groups of various chemical nature."> Хромопротеины - собирательное название сложных белков с окрашенными простетическими группами различной химической природы. гемопротеины (содержат гем), ретинальпротеины (содержат витамин А), флавопротеины (содержат витамин В2), кобамидпротеины (содержат витамин В12).!}
Ppt%5C34928-slozhnye_belki_ch1_33.jpg" alt=">Flavoproteins are enzymes of redox reactions. They contain derivatives of vitamin B2 flavin mononucleotide (FMN) and flavin adenine dinucleotide"> Флавопротеины - это ферменты окислительно-восстановительных реакций. Содержат производные витамина В2 флавинмононуклеотид (ФМН) и флавинадениндинуклеотид (ФАД). Связываются данные простетические группы ковалентно и придают желтое окрашивание. Эти простетические группы являются производными изоаллоксазина.!}
Ppt%5C34928-slozhnye_belki_ch1_34.jpg" alt=">Isoalloxazine is a heterocyclic compound, a derivative of pteridine. The isoalloxazine molecule consists of three aromatic rings -"> Изоаллоксазин - гетероциклическое соединения, производное птеридина. Молекула изоаллоксазина состоит из трех ароматических колец - бензольного, пиримидинового, пиразинового.!}
Ppt%5C34928-slozhnye_belki_ch1_35.jpg" alt=">Hemoproteins are heme-containing chromoproteins. Structurally similar iron or magnesium porphyrins are included as a non-protein component."> Гемопротеины - гем-содержащие хромопротеины. В качестве небелкового компонента включают структурно сходные железо- или магнийпорфирины. Белковый компонент может быть разнообразным как по составу, так и по структуре. Основу структуры простетической группы большинства гемосодержащих белков составляет порфириновое кольцо, являющееся в свою очередь производным тетрапиррольного соединения – порфирина. Порфирин!}
Ppt%5C34928-slozhnye_belki_ch1_36.jpg" alt=">The porphyrin ring is able to form coordination compounds with various metal ions. As a result of complex formation,"> Порфириновое кольцо способно образовывать координационные соединения с различными ионами металлов. В результате комплексообразования формируются металлопорфирины: содержащие ионы железа – гемоглобины, миоглобин, цитохромы, пероксидаза, каталаза и др. (красное окрашивание), содержщие ионы магния – хлорофилл (зеленое окрашивание). Витамин В12 (кобалимин) содержит координированный ион кобальта Со2+ в порфириноподобном макроцикле – коррине, состоящем из четырех частично гидрированных пиррольных колец (розовое окрашивание).!}
Ppt%5C34928-slozhnye_belki_ch1_37.jpg" alt=">Chlorophyll b. Chlorophyll is involved in photosynthesis.">!}
Ppt%5C34928-slozhnye_belki_ch1_38.jpg" alt="> Cytochromes differ in the amino acid composition of peptide chains, the number of chains and are divided into types a, b,"> Цитохромы различаются аминокислотным составом пептидных цепей, числом цепей и разделяются на типы а, b, с, d. Цитохромы находятся в составе дыхательной цепи и цепи микросомального окисления. Степень окисления железа в составе цитохромов меняется в отличие от гемоглобина и миоглобина Fe2+ ↔ Fe3+!}
Ppt%5C34928-slozhnye_belki_ch1_39.jpg" alt=">Myoglobin (Mb) is a protein found in red muscles, the main function of which is to create reserves"> Миоглобин (Мв) - белок, находящийся в красных мышцах, основная функция которого - создание запасов О2, необходимых при интенсивной мышечной работе. Мв - сложный белок, содержащий белковую часть - апоМв и небелковую часть - гем. Первичная структура апоМв определяет его компактную глобулярную конформацию и структуру активного центра, к которому присоединяется небелковая часть миоглобина - гем. Кислород, поступающий из крови в мышцы, связывается с Fe2+ гема в составе миоглобина. Мв - мономерный белок, имеющий очень высокое сродство к О2, поэтому отдача кислорода миоглобином происходит только при интенсивной мышечной работе, когда парциальное давление O2 резко снижается. Формирование пространственных структур и функционирование миоглобина.!}
Ppt%5C34928-slozhnye_belki_ch1_40.jpg" alt=">Formation of MB conformation."> Формирование конформации Мв. В красных мышцах на рибосомах в ходе трансляции идет синтез первичной структуры Мв, представленной специфической последовательностью 153 аминокислотных остатков. Вторичная структура Мв содержит восемь α-спиралей, называемых латинскими буквами от А до Н, между которыми имеются неспирализованные участки. Третичная структура Мв имеет вид компактной глобулы, в углублении которой между F и Е α-спиралями расположен активный центр.!}
Ppt%5C34928-slozhnye_belki_ch1_41.jpg" alt=">Myoglobin structure">!}
Ppt%5C34928-slozhnye_belki_ch1_42.jpg" alt=">Features of the structure and functioning of the Mv active center. The Mv active center is formed mainly by hydrophobic radicals"> Особенности строения и функционирования активного центра Мв. Активный центр Мв сформирован преимущественно гидрофобными радикалами аминокислот, далеко отстоящими друг от друга в первичной структуре (например, Три39 и Фен138). К активному центру присоединяется плохо растворимые в воде лиганды - гем и О2. Гем - специфический лиганд апоМв.!}
Ppt%5C34928-slozhnye_belki_ch1_43.jpg" alt=">Heme is based on four pyrrole rings connected by methenyl bridges; Fe2+ atom is located in the center,"> Основу гема составляют четыре пиррольных кольца, соединенных метенильными мостиками; в центре расположен атом Fe2+, соединенный с атомами азота пиррольных колец четырьмя координационными связями. В активном центре Мв кроме гидрофобных радикалов аминокислот имеются также остатки двух аминокислот с гидрофильными радикалами - Гис Е7 (Гис64) и Гис F8 (Гис93).!}
Ppt%5C34928-slozhnye_belki_ch1_44.jpg" alt=">His F8 forms a coordination bond with Fe2+ and firmly fixes the heme in the active site."> Гис F8 образует координационную связь с Fe2+ и прочно фиксирует гем в активном центре. Гис Е7 необходим для правильной ориентации в активном центре другого лиганда - O2 при его взаимодействии с Fe+2 гема. Микроокружение гема создает условия для прочного, но обратимого связывания O2 с Fe+2 и препятствует попаданию в гидрофобный активный центр воды, что может привести к его окислению в Fе3+.!}
Ppt%5C34928-slozhnye_belki_ch1_45.jpg" alt=">Oligomeric structure of Hb and regulation of Hb affinity for O2 by ligands. Human hemoglobins -"> Олигомерное строение Нв и регуляция сродства Нв к О2 лигандами. Гемоглобины человека - семейство белков, так же как и миоглобин относящиеся к сложным белкам (гемопротеинам). Они имеют тетрамерное строение и содержат две α-цепи, но различаются по строению двух других полипептидных цепей (2α-, 2х-цепи). Строение второй полипептидной цепи определяет особенности функционирования этих форм Нв. Около 98% гемоглобина эритроцитов взрослого человека составляет гемоглобин А (2α-, 2β-цепи). В период внутриутробного развития функционируют два основных типа гемоглобинов: эмбриональный Нв (2α, 2ε), который обнаруживается на ранних этапах развития плода, и гемоглобин F (фетальный) - (2α, 2γ), который приходит на смену раннему гемоглобину плода на шестом месяце внутриутробного развития и только после рождения замещается на Нв А.!}
Ppt%5C34928-slozhnye_belki_ch1_46.jpg" alt="> Hb A is a protein related to myoglobin (Mb) found in adult erythrocytes. Its structure"> Нв А - белок, родственный миоглобину (Мв), содержится в эритроцитах взрослого человека. Строение его отдельных протомеров аналогично таковому у миоглобина. Вторичная и третичная структуры миоглобина и протомеров гемоглобина очень сходны, несмотря на то что в первичной структуре их полипептидных цепей идентичны только 24 аминокислотных остатка (вторичная структура протомеров гемоглобина, так же как миоглобин, содержит восемь α-спиралей, обозначаемых латинскими буквами от А до Н, а третичная структура имеет вид компактной глобулы). Но в отличие от миоглобина гемоглобин имеет олигомерное строение, состоит из четырех полипептидных цепей, соединенных нековалентными связями.!}
Ppt%5C34928-slozhnye_belki_ch1_47.jpg" alt=">Oligomeric structure of hemoglobin">!}
Ppt%5C34928-slozhnye_belki_ch1_48.jpg" alt=">Each Hb protomer is connected to the non-protein part - the heme and neighboring protomers."> Каждый протомер Нв связан с небелковой частью - гемом и соседними протомерами. Соединение белковой части Нв с гемом аналогично таковому у миоглобина: в активном центре белка гидрофобные части гема окружены гидрофобными радикалами аминокислот за исключением Гис F8 и Гис Е7, которые расположены по обе стороны от плоскости гема и играют аналогичную роль в функционировании белка и связывании его с кислородом. Кроме того, Гис Е7 выполняет важную дополнительную роль в функционировании Нв. Свободный гем имеет в 25 000 раз более высокое сродство к СО, чем к О2. СО в небольших количествах образуется в организме и, учитывая его высокое сродство к гему, он мог бы нарушать транспорт необходимого для жизни клеток О2. Однако в составе гемоглобина сродство гема к оксиду углерода превышает сродство к О2 всего в 200 раз благодаря наличию в активном центре Гис Е7. Остаток этой аминокислоты создает !} optimal conditions for binding heme with O2 and weakens the interaction of heme with CO.
Ppt%5C34928-slozhnye_belki_ch1_49.jpg" alt=">">
Ppt%5C34928-slozhnye_belki_ch1_50.jpg" alt=">The heme pyrrole rings are located in the same plane, and the Fe2+ ion in the non-oxygenated state of Hb"> Пиррольные кольца гема расположены в одной плоскости, а ион Fe2+ в неоксигенированом состоянии Hb выступает над плоскостью на 0,6 А. При присоединении кислорода ион железа погружается в плоскость колец гема. В результате сдвигается и участок полипептидной цепи, нарушаются слабые связи в молекуле Hb и изменяется конформация всей глобулы. Таким образом, присоединение кислорода вызывает изменение пространственной структуры молекулы миоглобина или протомеров гемоглобина.!}
Ppt%5C34928-slozhnye_belki_ch1_51.jpg" alt=">Hemoglobin can exist both in free (deoxyhemoglobin) and in oxygenated form, attaching up to"> Гемоглобин может существовать как в свободной (дезоксигемоглобин), так и в оксигенированной форме, присоединяя до 4 молекул кислорода. Взаимодействие с кислородом 1-го протомера вызывает изменение его конформации, а также кооперативные конформационные изменения остальных протомеров. Сродство к кислороду возрастает, и присоединение О2 к активному центру 2-го протомера происходит легче, вызывая дальнейшую конформационную перестройку всей молекулы. В результате еще сильнее изменяется структура оставшихся протомеров и их активных центров, взаимодействие с О2 еще больше облегчается. В итоге 4-я молекула кислорода присоединяется к Hb примерно в 300 раз легче, чем 1-я. Так происходит в легких при высоком парциальном давлении кислорода.!}
Ppt%5C34928-slozhnye_belki_ch1_52.jpg" alt=">Cooperative changes in the conformation of the hemoglobin molecule upon interaction with oxygen">!}
Ppt%5C34928-slozhnye_belki_ch1_53.jpg" alt="> In tissues where the oxygen content is lower, on the contrary, the elimination of each O2 molecule facilitates the release of subsequent ones."> В тканях, где содержание кислорода ниже, наоборот, отщепление каждой молекулы О2 облегчает освобождение последующих. Таким образом, взаимодействие олигомерного белка гемоглобина с лигандом (О2) в одном центре связывания приводит к изменению конформации всей молекулы и других, пространственно удаленных центров, расположенных на других субъединицах (принцип «домино»). Подобные взаимосвязанные изменения структуры белка называют кооперативными конформационными изменениями. Они характерны для всех олигомерных белков и используются для регуляции их активности.!}
Ppt%5C34928-slozhnye_belki_ch1_54.jpg" alt=">The interaction of both proteins (Mb and Hb) with oxygen depends on its partial pressure in"> Взаимодействие обоих белков (Mb и Hb) с кислородом зависит от его парциального давления в тканях. Эта зависимость имеет разный характер, что связано с их особенностями структуры и функционирования. Гемоглобин имеет S-образную кривую насыщения, которая показывает, что субъединицы белка работают кооперативно, и чем больше кислорода они отдают, тем легче идет освобождение остальных молекул О2. Этот процесс зависит от изменения парциального давления кислорода в тканях. График насыщения миоглобина кислородом имеет характер простой гиперболы, т.е. насыщение Mb кислородом происходит быстро и отражает его функцию - обратимое связывание с кислородом, высвобождаемым гемоглобином, и освобождение в случае интенсивной физической нагрузки.!}
Ppt%5C34928-slozhnye_belki_ch1_55.jpg" alt=">Myoglobin and hemoglobin oxygen saturation curves">!}
Ppt%5C34928-slozhnye_belki_ch1_56.jpg" alt=">CO2 and H+, formed during the catabolism of organic substances, reduce the affinity of hemoglobin to O2 proportionally"> CO2 и Н+, образующиеся при катаболизме органических веществ, уменьшают сродство гемоглобина к О2 пропорционально их концентрации. Энергия, необходимая для работы клеток, вырабатывается преимущественно в митохондриях при окислении органических веществ с использованием O2, доставляемого из легких гемоглобином. В результате окисления органических веществ образуются конечные продукты их распада: СО2 и Н2O, количество которых пропорционально интенсивности протекающих процессов окисления. СO2 диффузией попадает из клеток в кровь и проникает в эритроциты, где под действием фермента карбоангидразы превращается в угольную кислоту. Эта слабая кислота диссоциирует на протон и бикарбонат ион. СО2 + Н2О → Н2СО3 → Н+ + НСО3-!}
Ppt%5C34928-slozhnye_belki_ch1_57.jpg" alt=">H+ ions are able to add to His146 radicals in hemoglobin β-chains, i.e. in areas remote from"> Ионы Н+ способны присоединятся к радикалам Гис146 в β-цепях гемоглобина, т.е. в участках, удаленных от гема. Протонирование гемоглобина снижает его сродство к О2, способствует отщеплению О2 от оксиНв, образованию дезоксиНв и увеличивает поступление кислорода в ткани пропорционально количеству образовавшихся протонов. Увеличение количества освобожденного кислорода в зависимости от увеличения концентрации Н+ в эритроцитах называется эффектом Бора (по имени датского физиолога Христиана Бора, впервые открывшего этот эффект). В легких высокое парциальное давление кислорода способствует его связыванию с дезоксиНв, что уменьшает сродство белка к Н+. Освободившиеся протоны под действием карбоангидразы взаимодействуют с бикарбонатами с образованием СО2 и Н2О!}
Ppt%5C34928-slozhnye_belki_ch1_58.jpg" alt=">Dependence of Hb affinity for O2 on CO2 and proton concentration (Bohr effect): A -"> Зависимость сродства Нв к О2 от концентрации СО2 и протонов (эффект Бора): А - влияние концентрации СО2 и Н+ на высвобождение О2 из комплекса с Нв (эффект Бора); Б - оксигенирование дезоксигемоглобина в легких, образование и выделение СО2.!}
Ppt%5C34928-slozhnye_belki_ch1_59.jpg" alt=">The resulting CO2 enters the alveolar space and is removed with exhaled air. Thus, the amount"> Образовавшийся СО2 поступает в альвеолярное пространство и удаляется с выдыхаемым воздухом. Таким образом, количество высвобождаемого гемоглобином кислорода в тканях регулируется продуктами катаболизма органических веществ: чем интенсивнее распад веществ, например при !} physical activity, the higher the concentration of CO2 and H+ and the more oxygen the tissues receive as a result of a decrease in the affinity of Hb for O2.
Ppt%5C34928-slozhnye_belki_ch1_60.jpg" alt=">A change in the functional activity of a protein when interacting with other ligands due to conformational changes is called allosteric"> Изменение функциональной активности белка при взаимодействии с другими лигандами вследствие конформационных изменений называется аллостерической регуляцией, а соединения-регуляторы - аллостерическими лигандами или эффекторами. Способность к аллостерической регуляции характерна, как правило, для олигомерных белков, т.е. для проявления аллостерического эффекта необходимо взаимодействие протомеров. При воздействии аллостерических лигандов белки меняют свою конформацию (в том числе и активного центра) и функцию.!}
Ppt%5C34928-slozhnye_belki_ch1_61.jpg" alt=">Allosteric regulation of Hb affinity for O2 by the 2,3-bis-phosphoglycerate ligand. In erythrocytes from the product"> Аллостерическая регуляция сродства Нв к О2 лигандом - 2,3-бис-фосфоглицератом. В эритроцитах из продукта окисления глюкозы - 1,3-бисфосфоглицерата синтезируется аллостерический лиганд гемоглобина - 2,3-бисфосфоглицерат (2,3-БФГ). В нормальных условиях концентрация 2,3-БФГ высокая и сравнима с концентрацией Нв. 2,3-БФГ имеет сильный отрицательный заряд (-5).!}
Ppt%5C34928-slozhnye_belki_ch1_62.jpg" alt=">There is a cavity in the center of the hemoglobin tetramer molecule. It is formed by the amino acid residues of all four protomers."> В центре тетрамерной молекулы гемоглобина находится полость. Ее образуют аминокислотные остатки всех четырех протомеров. В капиллярах тканей протонирование Нв (эффект Бора) приводит к разрыву связи между железом гема и О2. В молекуле дезоксигемоглобина по сравнению с оксигемоглобином возникают дополнительные ионные связи, соединяющие протомеры, вследствие чего размеры центральной полости по сравнению с оксигемоглобином увеличиваются. Центральная полость является местом присоединения 2,3-БФГ к гемоглобину. БФГ поступает в полость дезоксигемоглобина. 2,3-БФГ взаимодействует с гемоглобином в участке, удаленном от активных центров белка и относится к аллостерическим (регуляторным) лигандам, а центральная полость Нв является аллостерическим центром. 2,3-БФГ имеет сильный отрицательный заряд и взаимодействует с положительно заряженными группами двух β-цепей Нв. При этом его сродство к О2 снижается в 26 раз. В результате происходит высвобождение кислорода в капиллярах ткани при низком парциальном давлении О2. В легких высокое парциальное давление О2, наоборот, приводит к оксигенированию Нв и освобождению БФГ.!}
Ppt%5C34928-slozhnye_belki_ch1_63.jpg" alt=">BPG binding center is located in a positively charged cavity between 4 hemoglobin protomers. BPG interaction"> Центр связывания БФГ находится в положительно заряженной полости между 4 протомерами гемоглобина. Взаимодействие БФГ с центром связывания изменяет конформацию α- и β-протомеров НЬ и их активных центров. Сродство НЬ к молекулам О2 снижается и кислород высвобождается в ткани. В легких при высоком парциальном давлении О2 активные центры гемоглобина насыщаются за счет изменения конформации и БФГ вытесняется из аллостерического центра!}
Ppt%5C34928-slozhnye_belki_ch1_64.jpg" alt=">">
Ppt%5C34928-slozhnye_belki_ch1_65.jpg" alt=">Thus, oligomeric proteins have new properties compared to monomeric proteins. Attachment of ligands"> Таким образом, олигомерные белки обладают новыми по сравнению с мономерными белками свойствами. Присоединение лигандов на участках, пространственно удаленных друг от друга (аллостерических), способно вызывать конформационные изменения во всей белковой молекуле. Благодаря взаимодействию с регуляторными лигандами происходит изменение конформации и адаптация функции белковой молекулы к изменениям окружающей среды.!}
Ppt%5C34928-slozhnye_belki_ch1_66.jpg" alt=">About 15% carbon dioxide present in the blood is carried by hemoglobin molecules. In tissues, some of the molecules "> About 15% of the carbon dioxide present in the blood is carried by hemoglobin molecules. In tissues, some of the carbon dioxide molecules can attach to each protomer of the hemoglobin molecule, while the affinity of Hb for oxygen decreases. In the lungs, on the contrary, due to high partial pressure of oxygen, O2 binds to Hb and CO2 is released.
Ppt%5C34928-slozhnye_belki_ch1_67.jpg" alt=">">
Ppt%5C34928-slozhnye_belki_ch1_68.jpg" alt=">In the hemoglobin S molecule (as abnormal hemoglobin is called), 2 β-chains were mutated, in which"> В молекуле гемоглобина S (так назван аномальный гемоглобин) мутантными оказались 2 β-цепи, в которых глутамат, высокополярная отрицательно заряженная аминокислота в положении 6 была заменена валином, содержащим гидрофобный радикал.!}
The high specificity of protein binding to the ligand is provided by the complementarity of the structure of the active site of the protein with the structure of the ligand
Complementarity is understood as the spatial and chemical correspondence of interacting molecules. The ligand must be able to enter and spatially coincide with the conformation of the active site. This coincidence may not be complete, but due to the conformational lability of the protein, the active center is capable of small changes and is "adjusted" to the ligand. In addition, between the functional groups of the ligand and the amino acid radicals that form the active center, there should be bonds that hold the ligand in the active center. The bonds between the ligand and the active center of the protein can be either non-covalent (ionic, hydrogen, hydrophobic) or covalent.
1. Characteristics of the active center
The active center of a protein is a site relatively isolated from the environment surrounding the protein, formed by amino acid residues. In this area, each residue, due to its individual size and functional groups, forms the "relief" of the active center.
Combining such amino acids into a single functional complex changes the reactivity of their radicals, just as the sound changes musical instrument in the ensemble. Therefore, the amino acid residues that make up the active site are often referred to as an "ensemble" of amino acids.
The unique properties of the active center depend not only on the chemical properties of the amino acids that form it, but also on their exact mutual orientation in space. Therefore, even slight disturbances in the general conformation of a protein as a result of point changes in its primary structure or environmental conditions can lead to a change in the chemical and functional properties of the radicals that form the active center, disrupt the binding of the protein to the ligand and its function. During denaturation, the active center of proteins is destroyed, and their biological activity is lost.
The active center is often formed in such a way that the access of water to the functional groups of its radicals is limited; conditions are created for binding the ligand to amino acid radicals.
In some cases, the ligand is attached to only one of the atoms that has a certain reactivity, for example, the addition of O 2 to the iron of myoglobin or hemoglobin. However, the properties of a given atom to selectively interact with O 2 are determined by the properties of the radicals surrounding the iron atom in the composition of the topic. Heme is also found in other proteins, such as cytochromes. However, the function of the iron atom in cytochromes is different, it serves as an intermediary for the transfer of electrons from one substance to another, while iron becomes divalent or trivalent.
The main property of proteins underlying their functions is the selectivity of attaching specific ligands to certain parts of the protein molecule.
2. Variety of ligands
Ligands can be inorganic (often metal ions) and organic substances, low molecular weight and high molecular weight substances;
there are ligands that change their chemical structure when attached to the active center of the protein (substrate changes in the active center of the enzyme);
there are ligands that attach to the protein only at the moment of functioning (for example, O 2 transported by hemoglobin), and ligands that are constantly associated with the protein and play an auxiliary role in the functioning of proteins (for example, iron, which is part of hemoglobin).
In cases where the amino acid residues that form the active center cannot ensure the functioning of this protein, non-protein molecules can attach to certain parts of the active center. So, in the active center of many enzymes there is a metal ion (cofactor) or an organic non-protein molecule (coenzyme). The non-protein part, strongly associated with the active site of the protein and necessary for its functioning, is called the "prostatic group". Myoglobin, hemoglobin and cytochromes have a prosthetic group in the active center - heme containing iron.
The connection of protomers in an oligomeric protein is an example of the interaction of high molecular weight ligands. Each protomer connected to other protomers serves as a ligand for them, just as they are for it.
Sometimes the addition of a ligand changes the conformation of the protein, resulting in the formation of a binding site with other ligands. For example, the calmodulin protein, after binding to four Ca 2+ ions in specific areas, acquires the ability to interact with certain enzymes, changing their activity.
8. Quaternary structure of proteins. Features of the structure and functioning of oligomeric proteins on the example of hemoglobin. Cooperative changes in protomer conformation. Possibility of regulation of the biological function of oligomeric proteins by allosteric ligands.
Under the Quaternary structure is meant a way of laying in space individual polypeptide chains with the same (or different) primary, secondary or tertiary structure, and the formation of a single macromolecular formation in structural and functional respects. Many functional proteins consist of several polypeptide chains connected not by covalent bonds, but by non-covalent bonds (similar to those that ensure the stability of the tertiary structure). Each individual polypeptide chain, called a protomer, monomer or subunit, most often does not have biological activity. A protein acquires this ability with a certain way of spatial association of its constituent protomers, i.e. there is a new quality that is not characteristic of a monomeric protein. The resulting molecule is commonly called an oligomer (or multimer). Oligomeric proteins are often built from an even number of protomers (from 2 to 4, rarely from 6 to 8) with the same or different molecular weights - from several thousand to hundreds of thousands. In particular, the hemoglobin molecule consists of two identical α- and two β-polypeptide chains, i.e. is a tetramer.
Cooperative changes in protomer conformation.
A change in the conformation and, consequently, in the functional properties of all protomers of an oligomeric protein when a ligand is attached to only one of them is called cooperative changes in the conformation of the protomers.
Allosteric regulation . The enzyme changes activity through a non-covalently associated effector. The binding occurs in the area, spatially remote from the active (catalytic) site. This binding causes a conformational change in the protein molecule, leading to a change in the specific geometry of the catalytic site. Activity can increase - this is enzyme activation, or decrease - this is inhibition. The "message" about the attachment of the allosteric activator is transmitted through conformational changes to the catalytic subunit, which becomes complementary to the substrate, and the enzyme "turns on". When the activator is removed, the enzyme again goes into an inactive form and "turns off". Allosteric regulation is the main mode of regulation of metabolic pathways.
Protein modules (domains)
Usually, proteins formed by one polypeptide chain are a compact formation, each part of which cannot function and exist separately, retaining the same structure. However, in some cases, with a high content of amino acid residues (more than 200), not one, but several independent compact regions of one polypeptide chain are found in the three-dimensional structure. These fragments of the polypeptide chain, similar in properties to independent globular proteins, are called modules or domains . For example, there are two domains in dehydrogenases, one binds NAD + and this domain is similar in structure for all NAD-dependent dehydrogenases, and the other domain binds the substrate and differs in structure for different dehydrogenases.
Fatty acid synthase, which is a single polypeptide chain, has 7 domains to catalyze 7 reactions. It is assumed that the synthase domains were once combined into one protein as a result of gene fusion. The connection of modules (domains) into one protein contributes to the rapid emergence and evolution of new functional proteins.
The active site of a protein it is the binding site of the protein to the ligand. A site is formed on the surface of the globule, which can attach to itself other molecules called ligands . The active center of a protein is formed from side groups of amino acids that are close at the level of the tertiary structure. In the linear sequence of the peptide chain, they can be located at a distance significantly removed from each other. Proteins exhibit high specificity when interacting with a ligand. The high specificity of the interaction of the protein with the ligand is provided by the complementarity of the structure of the active site of the protein with the structure of the ligand. complementarity is the spatial and chemical correspondence of interacting molecules. Protein ligand binding sites are often located between domains (for example, a trypsin ligand binding site has 2 domains separated by a groove).
The functioning of proteins is based on their specific interaction with ligands. 50,000 individual proteins containing unique active centers that can bind only to specific ligands and, due to the structural features of the active center, exhibit their inherent functions. Obviously, the primary structure contains information about the function of proteins.
Quaternary structure- this is the highest level of structural organization, not possible for all proteins. Quaternary structure is understood as a way of laying polypeptide chains in space and the formation of a single macromolecular formation in structural and functional respects. Each individual polypeptide chain, called protomer or subunits , most often does not have biological activity. The protein acquires this ability with a certain way of spatial association of its constituent protomers. The resulting molecule is called oligomer (multimer) .
The quaternary structure is stabilized by non-covalent bonds that arise between the contact pads of protomers that interact with each other by the type of complementarity.
Proteins with a quaternary structure include many enzymes (lactate dehydrogenase, glutamate dehydrogenase, etc.), as well as hemoglobin, the muscle contractile protein myosin. Some proteins have a small number of subunits 2-8, others have hundreds or even thousands of subunits. For example, the tobacco mosaic virus protein has 2130 subunits.
A typical example of a protein having a quaternary structure is hemoglobin. The hemoglobin molecule consists of 4 subunits, i.e., polypeptide chains, each of which is associated with heme, of which 2 polypeptide chains are called -2afla and -2beta. They differ in the primary structure and length of the polypeptide chain.
The bonds forming the quaternary structure are less strong. Under the influence of some agents, the protein is separated into separate subunits. When the agent is removed, the subunits can reunite and the biological function of the protein is restored. So, when urea is added to a hemoglobin solution, it decomposes into 4 of its subunits, when urea is removed, the structural and functional role of hemoglobin is restored.
Module Structure | Themes |
Modular unit 1 | 1.1. Structural organization of proteins. Stages of formation of native conformation of proteins 1.2. Fundamentals of protein functioning. Drugs as ligands affecting protein function 1.3. Denaturation of proteins and the possibility of their spontaneous renativation |
Modular unit 2 | 1.4. Features of the structure and functioning of oligomeric proteins on the example of hemoglobin 1.5. Maintaining the native conformation of proteins in a cell 1.6. Variety of proteins. Protein families on the example of immunoglobulins 1.7. Physico-chemical properties of proteins and methods for their separation |
Modular unit 1 STRUCTURAL ORGANIZATION OF MONOMERIC PROTEINS AND THE BASIS OF THEIR FUNCTIONING
Learning objectives To be able to:
1. Use knowledge about the structural features of proteins and the dependence of protein functions on their structure to understand the mechanisms of development of hereditary and acquired proteinopathies.
2. Explain the mechanisms of the therapeutic action of certain drugs as ligands that interact with proteins and change their activity.
3. Use knowledge about the structure and conformational lability of proteins to understand their structural and functional instability and tendency to denaturation under changing conditions.
4. Explain the use of denaturing agents as means for sterilizing medical material and instruments, as well as as antiseptics.
Know:
1. Levels of structural organization of proteins.
2. The importance of the primary structure of proteins, which determines their structural and functional diversity.
3. The mechanism of formation of the active center in proteins and its specific interaction with the ligand, which underlies the functioning of proteins.
4. Examples of the influence of exogenous ligands (drugs, toxins, poisons) on the conformation and functional activity of proteins.
5. Causes and effects of protein denaturation, factors causing denaturation.
6. Examples of the use of denaturing factors in medicine as antiseptics and means for sterilizing medical instruments.
TOPIC 1.1. STRUCTURAL ORGANIZATION OF PROTEINS. STAGES FORMING A NATIVE
PROTEIN CONFORMATIONS
Proteins are polymeric molecules, the monomers of which are only 20 α-amino acids. The set and order of connection of amino acids in a protein is determined by the structure of genes in the DNA of individuals. Each protein, in accordance with its specific structure, performs its own function. The set of proteins of a given organism determines its phenotypic features, as well as the presence of hereditary diseases or a predisposition to their development.
1. Amino acids that make up proteins. peptide bond. Proteins are polymers built from monomers - 20 α-amino acids, the general formula of which is
Amino acids differ in structure, size, physicochemical properties of the radicals attached to the α-carbon atom. The functional groups of amino acids determine the features of the properties of different α-amino acids. The radicals found in α-amino acids can be divided into several groups:
proline, unlike the other 19 protein monomers, not an amino acid, but an imino acid, the radical in proline is associated with both the α-carbon atom and the imino group
Amino acids differ in their solubility in water. This is due to the ability of radicals to interact with water (to be hydrated).
TO hydrophilic include radicals containing anionic, cationic and polar uncharged functional groups.
TO hydrophobic include radicals containing methyl groups, aliphatic chains or cycles.
2. Peptide bonds link amino acids into peptides. During the synthesis of a peptide, the α-carboxyl group of one amino acid interacts with the α-amino group of another amino acid to form peptide bond:
Proteins are polypeptides, i.e. linear polymers of α-amino acids connected by a peptide bond (Fig. 1.1.)
Rice. 1.1. Terms used in describing the structure of peptides
The amino acid monomers that make up polypeptides are called amino acid residues. Chain of repeating groups - NH-CH-CO- forms peptide backbone. An amino acid residue having a free α-amino group is called N-terminal, and one having a free α-carboxyl group is called C-terminal. Peptides are written and read from the N-terminus to the C-terminus.
The peptide bond formed by the imino group of proline differs from other peptide bonds: the nitrogen atom of the peptide group lacks hydrogen,
instead, there is a bond with the radical, as a result, one side of the cycle is included in the peptide backbone:
Peptides differ in amino acid composition, the number of amino acids and the order of amino acids, for example, Ser-Ala-Glu-Gis and His-Glu-Ala-Ser are two different peptides.
Peptide bonds are very strong, and harsh conditions are required for their chemical non-enzymatic hydrolysis: the analyzed protein is hydrolyzed in concentrated hydrochloric acid at a temperature of about 110°C for 24 hours. In a living cell, peptide bonds can be broken by proteolytic enzymes, called proteases or peptide hydrolases.
3. Primary structure of proteins. Amino acid residues in the peptide chains of different proteins do not alternate randomly, but are arranged in a certain order. The linear sequence or sequence of amino acid residues in a polypeptide chain is called the primary structure of a protein.
The primary structure of each individual protein is encoded in a DNA molecule (in a region called a gene) and is realized during transcription (rewriting information on mRNA) and translation (synthesis of the protein's primary structure). Consequently, the primary structure of the proteins of an individual person is information inherited from parents to children that determines the structural features of the proteins of a given organism, on which the function of existing proteins depends (Fig. 1.2.).
Rice. 1.2. The relationship between the genotype and the conformation of proteins synthesized in the body of an individual
Each of the approximately 100,000 individual proteins in the human body has unique primary structure. Molecules of one type of protein (for example, albumin) have the same alternation of amino acid residues, which distinguishes albumin from any other individual protein.
The sequence of amino acid residues in the peptide chain can be considered as a form of information recording. This information determines the spatial folding of a linear peptide chain into a more compact three-dimensional structure called conformation squirrel. The process of formation of a functionally active protein conformation is called folding.
4. Conformation of proteins. Free rotation in the peptide backbone is possible between the nitrogen atom of the peptide group and the neighboring α-carbon atom, as well as between the α-carbon atom and the carbonyl group carbon. Due to the interaction of functional groups of amino acid residues, the primary structure of proteins can acquire more complex spatial structures. In globular proteins, two main levels of folding of the conformation of peptide chains are distinguished: secondary And tertiary structure.
Secondary structure of proteins- this is a spatial structure formed as a result of the formation of hydrogen bonds between the functional groups -C=O and -NH- of the peptide backbone. In this case, the peptide chain can acquire regular structures of two types: α-helices And β structures.
IN α-helices hydrogen bonds are formed between the oxygen atom of the carbonyl group and the hydrogen of the amide nitrogen of the 4th amino acid from it; side chains of amino acid residues
located along the periphery of the helix, not participating in the formation of the secondary structure (Fig. 1.3.).
Bulky radicals or radicals carrying the same charges prevent the formation of an α-helix. The proline residue, which has a ring structure, interrupts the α-helix, since due to the lack of hydrogen at the nitrogen atom in the peptide chain, it is impossible to form a hydrogen bond. The bond between nitrogen and the α-carbon atom is part of the proline cycle, so the peptide backbone acquires a bend in this place.
β-Structure is formed between the linear regions of the peptide backbone of one polypeptide chain, thus forming folded structures. Polypeptide chains or parts thereof can form parallel or antiparallel β-structures. In the first case, the N- and C-terminals of the interacting peptide chains coincide, and in the second case, they have the opposite direction (Fig. 1.4).
Rice. 1.3. Protein secondary structure - α-helix
Rice. 1.4. Parallel and antiparallel β-pleated structures
β-structures are indicated by wide arrows: A - Antiparallel β-structure. B - Parallel β-pleated structures
In some proteins, β-structures can be formed due to the formation of hydrogen bonds between the atoms of the peptide backbone of different polypeptide chains.
Also found in proteins areas with irregular secondary structure, which include bends, loops, turns of the polypeptide backbone. They are often located in places where the direction of the peptide chain changes, for example, during the formation of a parallel β-sheet structure.
By the presence of α-helices and β-structures, globular proteins can be divided into four categories.
Rice. 1.5. Secondary structure of myoglobin (A) and hemoglobin β-chain (B), containing eight α-helices
Rice. 1.6. Secondary structure of triose phosphate isomerase and pyruvate kinase domain
Rice. 1.7. Secondary structure of immunoglobulin constant domain (A) and superoxide dismutase enzyme (B)
IN fourth category included proteins that have in their composition a small amount of regular secondary structures. These proteins include small, cysteine-rich proteins or metalloproteins.
Tertiary structure of a protein- a type of conformation formed due to interactions between amino acid radicals, which can be located at a considerable distance from each other in the peptide chain. In this case, most proteins form a spatial structure resembling a globule (globular proteins).
Since the hydrophobic radicals of amino acids tend to combine with the help of the so-called hydrophobic interactions and intermolecular van der Waals forces, a dense hydrophobic core is formed inside the protein globule. Hydrophilic ionized and non-ionized radicals are mainly located on the surface of the protein and determine its solubility in water.
Rice. 1.8. Types of bonds that arise between amino acid radicals during the formation of the tertiary structure of a protein
1 - ionic bond- occurs between positively and negatively charged functional groups;
2 - hydrogen bond- occurs between the hydrophilic uncharged and any other hydrophilic group;
3 - hydrophobic interactions- occur between hydrophobic radicals;
4 - disulfide bond- is formed due to the oxidation of SH-groups of cysteine residues and their interaction with each other
Hydrophilic amino acid residues inside the hydrophobic core can interact with each other using ionic And hydrogen bonds(Fig. 1.8).
Ionic and hydrogen bonds, as well as hydrophobic interactions, are among the weak ones: their energy slightly exceeds the energy of the thermal motion of molecules at room temperature. Protein conformation is maintained by the occurrence of many such weak bonds. Since the atoms that make up the protein are in constant motion, it is possible to break some weak bonds and form others, which leads to small movements of individual sections of the polypeptide chain. This property of proteins to change conformation as a result of breaking some and forming other weak bonds is called conformational lability.
The human body has systems that support homeostasis- the constancy of the internal environment within certain limits acceptable for a healthy organism. Under conditions of homeostasis, small changes in conformation do not disrupt the overall structure and function of proteins. The functionally active conformation of a protein is called native conformation. A change in the internal environment (for example, the concentration of glucose, Ca ions, protons, etc.) leads to a change in the conformation and disruption of the functions of proteins.
The tertiary structure of some proteins is stabilized disulfide bonds, formed by the interaction of -SH groups of two residues
Rice. 1.9. The formation of a disulfide bond in a protein molecule
cysteine (Fig. 1.9). Most intracellular proteins do not have covalent disulfide bonds in their tertiary structure. Their presence is characteristic of proteins secreted by the cell, which ensures their greater stability in extracellular conditions. So, disulfide bonds are present in the molecules of insulin and immunoglobulins.
Insulin- a protein hormone synthesized in the β-cells of the pancreas and secreted into the blood in response to an increase in the concentration of glucose in the blood. In the structure of insulin, there are two disulfide bonds connecting the polypeptide A- and B-chains, and one disulfide bond inside the A-chain (Fig. 1.10).
Rice. 1.10. Disulfide bonds in the structure of insulin
5. Super secondary structure of proteins. In proteins different in primary structure and functions, sometimes similar combinations and interposition of secondary structures, which are called the supersecondary structure. It occupies an intermediate position between secondary and tertiary structures, since it is a specific combination of secondary structure elements during the formation of the tertiary structure of a protein. Supersecondary structures have specific names such as "α-helix-turn-a-helix", "leucine zipper", "zinc fingers", etc. Such supersecondary structures are characteristic of DNA-binding proteins.
"Leucine zipper". This kind of super secondary structure is used to connect two proteins. On the surface of interacting proteins there are α-helical regions containing at least four leucine residues. Leucine residues in the α-helix are located six amino acids apart from each other. Since each turn of the α-helix contains 3.6 amino acid residues, leucine radicals are found on the surface of every other turn. The leucine residues of the α-helix of one protein can interact with the leucine residues of another protein (hydrophobic interactions), connecting them together (Fig. 1.11.). Many DNA-binding proteins function as part of oligomeric complexes, where individual subunits are linked to each other by "leucine zippers".
Rice. 1.11. "Leucine zipper" between α-helical regions of two proteins
Histones are an example of such proteins. Histones- nuclear proteins, which include a large number of positively charged amino acids - arginine and lysine (up to 80%). Histone molecules are combined into oligomeric complexes containing eight monomers with the help of "leucine fasteners", despite the significant homonymous charge of these molecules.
"Zinc Finger"- a variant of the supersecondary structure, characteristic of DNA-binding proteins, has the form of an elongated fragment on the surface of the protein and contains about 20 amino acid residues (Fig. 1.12). The shape of the "stretched finger" is supported by a zinc atom associated with four amino acid radicals - two cysteine residues and two histidine residues. In some cases, instead of histidine residues, there are cysteine residues. The two closely spaced cysteine residues are separated from the other two Gisili residues by a Cys sequence of approximately 12 amino acid residues. This region of the protein forms an α-helix, the radicals of which can specifically bind to the regulatory regions of the DNA major groove. The specificity of the binding of an individual
Rice. 1.12. The primary structure of a section of DNA-binding proteins that form the “zinc finger” structure (letters indicate the amino acids that make up this structure)
regulatory DNA-binding protein depends on the sequence of amino acid residues located in the "zinc finger". Such structures contain, in particular, receptors for steroid hormones involved in the regulation of transcription (reading information from DNA to RNA).
TOPIC 1.2. BASES OF PROTEIN FUNCTIONING. DRUGS AS LIGANDS AFFECTING PROTEIN FUNCTION
1. The active center of the protein and its interaction with the ligand. During the formation of the tertiary structure, on the surface of a functionally active protein, usually in a recess, a site is formed formed by amino acid radicals that are far apart in the primary structure. This site, which has a unique structure for a given protein and is able to specifically interact with a certain molecule or group of similar molecules, is called the protein binding site with a ligand or active site. Ligands are molecules that interact with proteins.
High specificity The interaction of the protein with the ligand is ensured by the complementarity of the structure of the active center with the structure of the ligand.
complementarity is the spatial and chemical correspondence of the interacting surfaces. The active center must not only spatially correspond to the ligand included in it, but also between the functional groups of the radicals included in the active center and the ligand, bonds must be formed (ionic, hydrogen, and hydrophobic interactions) that keep the ligand in the active center (Fig. 1.13 ).
Rice. 1.13. Complementary interaction of a protein with a ligand
Some ligands, when attached to the active center of a protein, play an auxiliary role in the functioning of proteins. Such ligands are called cofactors, and proteins that have a non-protein part in their composition are called complex proteins(in contrast to simple proteins, consisting only of the protein part). The non-protein part that is firmly attached to the protein is called prosthetic group. For example, the composition of myoglobin, hemoglobin and cytochromes contains a prosthetic group firmly attached to the active center - a heme containing an iron ion. Complex proteins containing heme are called hemoproteins.
When specific ligands are attached to proteins, the function of these proteins is manifested. Thus, albumin, the most important protein in blood plasma, exhibits its transport function by attaching hydrophobic ligands to the active center, such as fatty acids, bilirubin, some drugs, etc. (Fig. 1.14)
Ligands interacting with the three-dimensional structure of the peptide chain can be not only low-molecular organic and inorganic molecules, but also macromolecules:
DNA (examples discussed above with DNA-binding proteins);
Polysaccharides;
Rice. 1.14. Relationship between genotype and phenotype
The unique primary structure of human proteins, encoded in the DNA molecule, is realized in cells in the form of a unique conformation, active site structure, and protein functions.
In these cases, the protein recognizes a specific region of the ligand that is commensurate with and complementary to the binding site. So on the surface of hepatocytes there are receptor proteins for the hormone insulin, which also has a protein structure. The interaction of insulin with the receptor causes a change in its conformation and activation of signaling systems, leading to the accumulation of nutrients in hepatocytes after eating.
Thus, The functioning of proteins is based on the specific interaction of the active center of the protein with the ligand.
2. Domain structure and its role in the functioning of proteins. Long polypeptide chains of globular proteins often fold into several compact, relatively independent regions. They have an independent tertiary structure, resembling that of globular proteins, and are called domains. Due to the domain structure of proteins, their tertiary structure is easier to form.
In domain proteins, ligand binding sites are often located between domains. So, trypsin is a proteolytic enzyme that is produced by the exocrine part of the pancreas and is necessary for the digestion of food proteins. It has a two-domain structure, and the binding site of trypsin with its ligand - food protein - is located in the groove between the two domains. In the active center, the conditions necessary for the effective binding of a specific site of the food protein and the hydrolysis of its peptide bonds are created.
Different domains in a protein can move relative to each other when the active center interacts with the ligand (Fig. 1.15).
Hexokinase- an enzyme that catalyzes the phosphorylation of glucose with the help of ATP. The active site of the enzyme is located in the cleft between the two domains. When hexokinase binds to glucose, the surrounding domains close and the substrate is trapped, where phosphorylation occurs (see Fig. 1.15).
Rice. 1.15. Binding of hexokinase domains to glucose
In some proteins, domains perform independent functions by binding to various ligands. Such proteins are called multifunctional.
3. Drugs - ligands that affect the function of proteins. The interaction of proteins with ligands is specific. However, due to the conformational lability of the protein and its active site, it is possible to choose another substance that could also interact with the protein in the active site or another part of the molecule.
A substance that is similar in structure to a natural ligand is called structural analogue of the ligand or an unnatural ligand. It also interacts with a protein in the active site. A structural analog of a ligand can both enhance protein function (agonist) and reduce it (antagonist). The ligand and its structural analogs compete with each other for protein binding at the same site. Such substances are called competitive modulators(regulators) of protein functions. Many drugs act as protein inhibitors. Some of them are obtained by chemical modification of natural ligands. Protein function inhibitors can be drugs and poisons.
Atropine is a competitive inhibitor of M-cholinergic receptors. Acetylcholine is a neurotransmitter for the transmission of nerve impulses through cholinergic synapses. To conduct excitation, acetylcholine released into the synaptic cleft must interact with the protein - the receptor of the postsynaptic membrane. Two types found cholinergic receptors:
M-receptor in addition to acetylcholine, it selectively interacts with muscarine (fly agaric toxin). M - cholinergic receptors are present on smooth muscles and, when interacting with acetylcholine, cause their contraction;
H-receptor binds specifically to nicotine. N-cholinergic receptors are found in the synapses of striated skeletal muscles.
specific inhibitor M-cholinergic receptors is atropine. It is found in belladonna and henbane plants.
Atropine has functional groups and their spatial arrangement similar to acetylcholine in its structure, therefore it belongs to competitive inhibitors of M-cholinergic receptors. Given that the binding of acetylcholine to M-cholinergic receptors causes contraction of smooth muscles, atropine is used as a drug that relieves their spasm. (antispasmodic). Thus, it is known the use of atropine to relax the eye muscles when viewing the fundus, as well as to relieve spasms in gastrointestinal colic. M-cholinergic receptors are also present in the central nervous system(CNS), so large doses of atropine can cause unwanted reaction from the side of the central nervous system: motor and mental agitation, hallucinations, convulsions.
Ditilin is a competitive agonist of H-cholinergic receptors that inhibits the function of neuromuscular synapses.
The neuromuscular synapses of skeletal muscles contain H-cholinergic receptors. Their interaction with acetylcholine leads to muscle contractions. In some surgical operations, as well as in endoscopic studies, drugs are used that cause relaxation of skeletal muscles. (muscle relaxants). These include dithylin, which is a structural analogue of acetylcholine. It attaches to H-cholinergic receptors, but unlike acetylcholine, it is very slowly destroyed by the enzyme acetylcholinesterase. As a result of the prolonged opening of ion channels and persistent depolarization of the membrane, the conduction of the nerve impulse is disrupted and muscle relaxation occurs. Initially, these properties were found in curare poison, therefore such drugs are called curariform.
TOPIC 1.3. PROTEIN DENATURATION AND THE POSSIBILITY OF THEIR SPONTANEOUS RENATIVATION
1. Since the native conformation of proteins is maintained due to weak interactions, changes in the composition and properties of the environment surrounding the protein, the impact chemical reagents and physical factors cause a change in their conformation (property of conformational lability). The rupture of a large number of bonds leads to the destruction of the native conformation and protein denaturation.
Protein denaturation- this is the destruction of their native conformation under the action of denaturing agents, caused by the breaking of weak bonds that stabilize the spatial structure of the protein. Denaturation is accompanied by the destruction of the unique three-dimensional structure and active center of the protein and the loss of its biological activity (Fig. 1.16).
All denatured molecules of one protein acquire a random conformation that differs from other molecules of the same protein. The amino acid radicals that form the active center turn out to be spatially distant from each other, i.e. the specific binding site of the protein with the ligand is destroyed. During denaturation, the primary structure of proteins remains unchanged.
The use of denaturing agents in biological research and medicine. In biochemical studies, before the determination of low molecular weight compounds in a biological material, proteins are usually removed from the solution first. For this purpose, trichloroacetic acid (TCA) is most often used. After adding TCA to the solution, denatured proteins precipitate and are easily removed by filtration (Table 1.1.)
In medicine, denaturing agents are often used to sterilize medical instruments and material in autoclaves (denaturing agent - high temperature) and as antiseptics (alcohol, phenol, chloramine) to treat contaminated surfaces containing pathogenic microflora.
2. Spontaneous protein regeneration- proof of the determinism of the primary structure, conformation and function of proteins. Individual proteins are products of one gene that have an identical amino acid sequence and acquire the same conformation in the cell. The fundamental conclusion that the primary structure of a protein already contains information about its conformation and function was made on the basis of the ability of some proteins (in particular, ribonuclease and myoglobin) to spontaneous renativation - the restoration of their native conformation after denaturation.
The formation of spatial protein structures is carried out by the method of self-assembly - a spontaneous process in which the polypeptide chain, which has a unique primary structure, tends to adopt a conformation with the lowest free energy in solution. The ability to regenerate proteins that retain their primary structure after denaturation was described in an experiment with the enzyme ribonuclease.
Ribonuclease is an enzyme that breaks bonds between individual nucleotides in an RNA molecule. This globular protein has one polypeptide chain, the tertiary structure of which is stabilized by many weak and four disulfide bonds.
Treatment of ribonuclease with urea, which breaks hydrogen bonds in the molecule, and a reducing agent, which breaks disulfide bonds, leads to enzyme denaturation and loss of its activity.
Removal of denaturing agents by dialysis leads to restoration of protein conformation and function, i.e. to reanimation. (Fig. 1.17).
Rice. 1.17. Denaturation and renativation of ribonuclease
A - native conformation of ribonuclease, in the tertiary structure of which there are four disulfide bonds; B - denatured ribonuclease molecule;
B - renative ribonuclease molecule with restored structure and function
1. Complete table 1.2.
Table 1.2. Classification of amino acids according to the polarity of radicals
2. Write the formula of a tetrapeptide:
Asp - Pro - Fen - Liz
a) isolate the repeating groups in the peptide that form the peptide backbone and the variable groups represented by amino acid radicals;
b) designate the N- and C-termini;
c) underline the peptide bonds;
d) write another peptide consisting of the same amino acids;
e) count the number options tetrapeptide with the same amino acid composition.
3. Explain the role of the primary structure of proteins on the example of a comparative analysis of two structurally similar and evolutionarily close peptide hormones of the mammalian neurohypophysis - oxytocin and vasopressin (Table 1.3).
Table 1.3. Structure and function of oxytocin and vasopressin
For this:
a) compare the composition and amino acid sequence of the two peptides;
b) find the similarity of the primary structure of the two peptides and the similarity of their biological action;
c) find the differences in the structure of the two peptides and the difference in their functions;
d) draw a conclusion about the influence of the primary structure of peptides on their functions.
4. Describe the main stages in the formation of the conformation of globular proteins (secondary, tertiary structures, the concept of a supersecondary structure). Specify the types of bonds involved in the formation of protein structures. Which amino acid radicals can participate in the formation of hydrophobic interactions, ionic, hydrogen bonds.
Give examples.
5. Define the concept of "conformational lability of proteins", indicate the reasons for its existence and significance.
6. Explain the meaning of the following phrase: “Proteins function based on their specific interaction with a ligand”, using terms and explaining their meaning: protein conformation, active site, ligand, complementarity, protein function.
7. Using one of the examples, explain what domains are and what their role is in the functioning of proteins.
TASKS FOR SELF-CONTROL
1. Set a match.
Functional group in the amino acid radical:
A. Carboxyl group B. Hydroxyl group C Guanidine group D. Thiol group E. Amino group
2. Choose the correct answers.
Amino acids with polar uncharged radicals are:
A. Tsis B. Asn
B. Glu G. Three
3. Choose the correct answers.
Amino acid radicals:
A. Provide specificity of the primary structure B. Participate in the formation of the tertiary structure
B. Being located on the surface of the protein, they affect its solubility D. Form an active center
D. Participate in the formation of peptide bonds
4. Choose the correct answers.
Hydrophobic interactions can form between amino acid radicals:
A. Tre Lay B. Pro Three
B. Met Ile G. Tir Ala D. Val Fen
5. Choose the correct answers.
Ionic bonds can form between amino acid radicals:
A. Gln Asp B. Apr Liz
B. Liz Glu G. Geese Asp D. Asn Apr
6. Choose the correct answers.
Hydrogen bonds can form between amino acid radicals:
A. Ser Gln B. Cis Tre
B. Asp Liz G. Glu Asp D. Asn Tre
7. Set a match.
The type of bond involved in the formation of the protein structure:
A. Primary structure B. Secondary structure
B. Tertiary structure
D. Supersecondary structure E. Conformation.
1. Hydrogen bonds between the atoms of the peptide backbone
2. Weak bonds between functional groups of amino acid radicals
3. Bonds between α-amino and α-carboxyl groups of amino acids
8. Choose the correct answers. Trypsin:
A. Proteolytic enzyme B. Contains two domains
B. Hydrolyzes starch
D. The active center is located between domains. D. Consists of two polypeptide chains.
9. Choose the correct answers. Atropine:
A. Neurotransmitter
B. Structural analogue of acetylcholine
B. Interacts with H-cholinergic receptors
G. Enhances the conduction of a nerve impulse through cholinergic synapses
D. Competitive inhibitor of M-cholinergic receptors
10. Choose the correct statements. In proteins:
A. The primary structure contains information about the structure of its active site
B. The active center is formed at the level of the primary structure
B. Conformation is rigidly fixed by covalent bonds
D. The active site can interact with a group of similar ligands
due to the conformational lability of proteins D. Changing the environment can affect the affinity of the active
center to ligand
1. 1-C, 2-D, 3-B.
3. A, B, C, D.
7. 1-B, 2-D, 3-A.
8. A, B, C, D.
BASIC TERMS AND CONCEPTS
1. Protein, polypeptide, amino acids
2. Primary, secondary, tertiary protein structures
3. Conformation, native protein conformation
4. Covalent and weak bonds in a protein
5. Conformational lability
6. Protein active site
7. Ligands
8. Protein folding
9. Structural analogues of ligands
10. Domain proteins
11. Simple and complex proteins
12. Protein denaturation, denaturing agents
13. Protein regeneration
Solve problems
"Structural organization of proteins and the basis of their functioning"
1. The main function of the protein - hemoglobin A (HbA) - is the transport of oxygen to the tissues. In the human population, multiple forms of this protein with altered properties and function are known - the so-called abnormal hemoglobins. For example, hemoglobin S found in the erythrocytes of patients with sickle cell anemia (HbS) has been found to have low solubility under conditions of low oxygen partial pressure (as occurs in venous blood). This leads to the formation of aggregates of this protein. The protein loses its function, precipitates, and the red blood cells become irregular (some of them form a sickle shape) and are destroyed faster than usual in the spleen. As a result, sickle cell anemia develops.
The only difference in the primary structure of HvA was found in the N-terminal region of the β-chain of hemoglobin. Compare the N-terminal regions of the β-chain and show how changes in the primary structure of a protein affect its properties and functions.
For this:
a) write the amino acid formulas by which HvA differ and compare the properties of these amino acids (polarity, charge).
b) draw a conclusion about the reason for the decrease in solubility and the violation of oxygen transport in the tissue.
2. The figure shows a diagram of the structure of a protein that has a ligand-binding center (active center). Explain why a protein is selective in choosing a ligand. For this:
a) remember what the active center of the protein is, and consider the structure of the active center of the protein shown in the figure;
b) write the formulas of the amino acid radicals that make up the active center;
c) draw a ligand that could specifically interact with the active site of the protein. Indicate on it the functional groups capable of forming bonds with the amino acid radicals that make up the active center;
d) indicate the types of bonds that arise between the ligand and the amino acid radicals of the active center;
e) Explain the basis for the specificity of the interaction of a protein with a ligand.
3.
The figure shows the active site of the protein and several ligands.
Determine which of the ligands is most likely to interact with the active site of the protein and why.
What types of bonds arise during the formation of the protein-ligand complex?
4. Structural analogs of natural protein ligands can be used as drugs to change the activity of proteins.
Acetylcholine is a mediator of excitation transmission in neuromuscular synapses. When acetylcholine interacts with proteins - receptors of the postsynaptic membrane of skeletal muscles, ion channels open and muscle contraction occurs. Dithylin is a drug used in some operations to relax the muscles, as it disrupts the transmission of nerve impulses through neuromuscular synapses. Explain the mechanism of action of dithylin as a muscle relaxant drug. For this:
a) write the formulas of acetylcholine and dithyline and compare their structures;
b) describe the mechanism of the relaxing action of dithylin.
5. In some diseases, the patient's body temperature rises, which is considered as a protective reaction of the body. However, high temperatures are detrimental to body proteins. Explain why at temperatures above 40 °C the function of proteins is disrupted and a threat to human life arises. To do this, remember:
1) The structure of proteins and the bonds that hold its structure in the native conformation;
2) How does the structure and function of proteins change with increasing temperature?;
3) What is homeostasis and why is it important to maintain human health.
Modular unit 2 OLIGOMERIC PROTEINS AS TARGETS FOR REGULATORY INFLUENCE. STRUCTURAL AND FUNCTIONAL VARIETY OF PROTEINS. PROTEIN SEPARATION AND PURIFICATION METHODS
Learning objectives To be able to:
1. Use knowledge about the features of the structure and functions of oligomeric proteins to understand the adaptive mechanisms of regulation of their functions.
2. Explain the role of chaperones in the synthesis and maintenance of protein conformation in a cell.
3. To explain the diversity of manifestations of life by the diversity of structures and functions of proteins synthesized in the body.
4. Analyze the relationship between the structure of proteins and their function by comparing related hemoproteins - myoglobin and hemoglobin, as well as representatives of five classes of proteins of the immunoglobulin family.
5. Apply knowledge about the features of the physicochemical properties of proteins to select methods for their purification from other proteins and impurities.
6. Interpret the results of the quantitative and qualitative composition of blood plasma proteins to confirm or clarify the clinical diagnosis.
Know:
1. Features of the structure of oligomeric proteins and adaptive mechanisms of regulation of their functions on the example of hemoglobin.
2. The structure and functions of chaperones and their importance for maintaining the native conformation of proteins in a cell.
3. Principles of grouping proteins into families according to the similarity of their conformation and functions on the example of immunoglobulins.
4. Methods for the separation of proteins based on the features of their physicochemical properties.
5. Electrophoresis of blood plasma as a method for assessing the qualitative and quantitative composition of proteins.
TOPIC 1.4. FEATURES OF THE STRUCTURE AND FUNCTIONING OF OLIGOMERIC PROTEINS ON THE EXAMPLE OF HEMOGLOBIN
1. Many proteins contain several polypeptide chains. Such proteins are called oligomeric, and individual circuits protomers. Protomers in oligomeric proteins are connected by many weak non-covalent bonds (hydrophobic, ionic, hydrogen). Interaction
protomers is carried out thanks to complementarity their contact surfaces.
The number of protomers in oligomeric proteins can vary greatly: hemoglobin contains 4 protomers, the enzyme aspartate aminotransferase - 12 protomers, and the protein of the tobacco mosaic virus includes 2120 protomers connected by non-covalent bonds. Therefore, oligomeric proteins can have very high molecular weights.
The interaction of one protomer with others can be considered as a special case of the interaction of a protein with a ligand, since each protomer serves as a ligand for other protomers. The number and method of connection of protomers in a protein is called quaternary protein structure.
Proteins can contain protomers of the same or different structure, for example, homodimers are proteins containing two identical protomers, and heterodimers are proteins containing two different protomers.
If proteins contain different protomers, then binding centers with different ligands that differ in structure can form on them. When the ligand binds to the active center, the function of this protein is manifested. A center located on a different protomer is called allosteric (other than active). Contacting allosteric ligand or effector, it performs a regulatory function (Fig. 1.18). The interaction of the allosteric center with the effector causes conformational changes in the structure of the entire oligomeric protein due to its conformational lability. This affects the affinity of the active site for a specific ligand and regulates the function of that protein. A change in the conformation and function of all protomers during the interaction of an oligomeric protein with at least one ligand is called cooperative conformation change. Effectors that enhance protein function are called activators and effectors that depress its function - inhibitors.
Thus, in oligomeric proteins, as well as proteins having a domain structure, a new property appears in comparison with monomeric proteins - the ability to allosteric regulation of functions (regulation by attaching different ligands to the protein). This can be seen by comparing the structures and functions of the two closely related complex proteins myoglobin and hemoglobin.
Rice. 1.18. Diagram of the structure of a dimeric protein
2. Formation of spatial structures and functioning of myoglobin.
Myoglobin (Mb) is a protein found in red muscles, the main function of which is the creation of O 2 reserves necessary for intense muscular work. MB is a complex protein containing a protein part - apoMB and a non-protein part - heme. The primary structure of apoMB determines its compact globular conformation and the structure of the active center, to which the non-protein part of myoglobin, heme, is attached. Oxygen from the blood to the muscles binds to Fe + 2 heme in the composition of myoglobin. MB is a monomeric protein with a very high affinity for O 2, therefore, oxygen is released by myoglobin only during intense muscular work, when the partial pressure of O 2 decreases sharply.
Formation of conformation MB. In red muscles, on ribosomes during translation, the synthesis of the primary structure of MB, represented by a specific sequence of 153 amino acid residues, takes place. The secondary structure of Mv contains eight α-helices, called Latin letters from A to H, between which there are non-spiralized sections. The tertiary structure of Mv has the form of a compact globule, in the recess of which, between the F and E α-helices, there is an active center (Fig. 1.19).
Rice. 1.19. Structure of myoglobin
3. Features of the structure and functioning of the MV active center. The active center of Mv is formed mainly by hydrophobic amino acid radicals that are far apart from each other in the primary structure (for example, Tri 3 9 and Phen 138) The ligands poorly soluble in water, heme and O 2, are attached to the active center. Heme is a specific apoMv ligand (Fig. 1.20), which is based on four pyrrole rings connected by methenyl bridges; in the center, there is an Fe+ 2 atom connected to the nitrogen atoms of the pyrrole rings by four coordination bonds. In addition to the hydrophobic radicals of amino acids, the active center of Mv also contains residues of two amino acids with hydrophilic radicals - Gis E 7(Gis 64) and Gis F 8(His 93) (Fig. 1.21).
Rice. 1.20. The structure of heme - the non-protein part of myoglobin and hemoglobin
Rice. 1.21. Location of heme and O 2 in the active site of apomyoglobin and hemoglobin protomers
Heme is covalently bonded to His F 8 via an iron atom. O 2 attaches to iron on the other side of the heme plane. His E 7 is necessary for the correct orientation of O 2 and facilitates the addition of oxygen to Fe + 2 heme
Gis F 8 forms a coordination bond with Fe+ 2 and firmly fixes heme in the active site. Gis E 7 is necessary for the correct orientation in the active center of another ligand - O 2 during its interaction with Fe + 2 heme. The heme microenvironment creates conditions for strong but reversible binding of O 2 with Fe + 2 and prevents water from entering the hydrophobic active site, which can lead to its oxidation to Fe + 3 .
The monomeric structure of MB and its active center determines the high affinity of the protein for O 2 .
4. Oligomeric structure of Hb and regulation of Hb affinity for O 2 by ligands. Human hemoglobins- a family of proteins, as well as myoglobin related to complex proteins (hemoproteins). They have a tetrameric structure and contain two α-chains, but differ in the structure of the other two polypeptide chains (2α-, 2x-chains). The structure of the second polypeptide chain determines the features of the functioning of these forms of Hb. About 98% of the hemoglobin in adult erythrocytes is hemoglobin A(2α-, 2p-chains).
During fetal development, there are two main types of hemoglobins: embryonic HB(2α, 2ε), which is found in the early stages of fetal development, and hemoglobin F (fetal)- (2α, 2γ), which replaces early fetal hemoglobin in the sixth month of fetal development and is replaced by Hb A only after birth.
Hv A is a protein related to myoglobin (Mv) found in adult erythrocytes. The structure of its individual protomers is similar to that of myoglobin. The secondary and tertiary structures of myoglobin and hemoglobin protomers are very similar, despite the fact that only 24 amino acid residues are identical in the primary structure of their polypeptide chains (the secondary structure of hemoglobin protomers, like myoglobin, contains eight α-helices, denoted by Latin letters from A to H , and the tertiary structure has the form of a compact globule). But unlike myoglobin, hemoglobin has an oligomeric structure, consists of four polypeptide chains connected by non-covalent bonds (Figure 1.22).
Each Hb protomer is associated with a non-protein part - heme and neighboring protomers. The connection of the protein part of Hb with heme is similar to that of myoglobin: in the active center of the protein, the hydrophobic parts of the heme are surrounded by hydrophobic amino acid radicals, with the exception of His F 8 and His E 7 , which are located on both sides of the heme plane and play a similar role in the functioning of the protein and its binding with oxygen (see the structure of myoglobin).
Rice. 1.22. Oligomeric structure of hemoglobin
Besides, Gis E 7 performs an important additional role in the functioning of NV. Free heme has a 25,000 times higher affinity for CO than for O 2 . CO is formed in small amounts in the body and, given its high affinity for heme, it could disrupt the transport of O 2 necessary for cell life. However, in the composition of hemoglobin, the affinity of heme for carbon monoxide exceeds the affinity for O 2 by only 200 times due to the presence of E 7 in the active center of His. The residue of this amino acid creates optimal conditions for the binding of heme to O2 and weakens the interaction of heme with CO.
5. The main function of Hb is the transport of O 2 from the lungs to the tissues. Unlike monomeric myoglobin, which has a very high affinity for O 2 and performs the function of storing oxygen in red muscles, the oligomeric structure of hemoglobin provides:
1) rapid saturation of Hb with oxygen in the lungs;
2) the ability of Hb to release oxygen in the tissues at a relatively high partial pressure of O 2 (20-40 mm Hg);
3) the possibility of regulating the affinity of Hb to O 2 .
6. Cooperative changes in the conformation of hemoglobin protomers accelerate the binding of O 2 in the lungs and its return to the tissues. In the lungs, a high partial pressure of O2 promotes its binding to Hb in the active site of four protomers (2α and 2β). The active center of each protomer, as in myoglobin, is located between two α-helices (F and E) in a hydrophobic pocket. It contains a non-protein part - heme, attached to the protein part by many weak hydrophobic interactions and one strong bond between Fe 2 + heme and His F 8 (see Fig. 1.21).
In deoxyhemoglobin, due to this connection with His F 8 , the Fe 2 + atom protrudes from the heme plane towards histidine. The binding of O 2 to Fe 2 + occurs on the other side of the heme in the His E 7 region with the help of a single free coordination bond. His E 7 provides optimal conditions for the binding of O 2 with heme iron.
The addition of O 2 to the Fe +2 atom of one protomer causes it to move into the heme plane, and behind it the histidine residue associated with it
Rice. 1.23. Change in the conformation of the hemoglobin protomer when combined with O 2
This leads to a change in the conformation of all polypeptide chains due to their conformational lability. Changing the conformation of other chains facilitates their interaction with the next O 2 molecules.
The fourth O 2 molecule attaches to hemoglobin 300 times easier than the first (Fig. 1.24).
Rice. 1.24. Cooperative changes in the conformation of hemoglobin protomers during its interaction with O 2
In tissues, each subsequent O 2 molecule is more easily cleaved off than the previous one, also due to cooperative changes in protomer conformation.
7. CO 2 and H +, formed during the catabolism of organic substances, reduce the affinity of hemoglobin for O 2 in proportion to their concentration. The energy necessary for cell functioning is produced mainly in mitochondria during the oxidation of organic substances using O 2 delivered from the lungs by hemoglobin. As a result of the oxidation of organic substances, the final products of their decay are formed: CO 2 and K 2 O, the amount of which is proportional to the intensity of the ongoing oxidation processes.
CO 2 diffuses from cells into the blood and penetrates into erythrocytes, where, under the action of the enzyme carbanhydrase, it turns into carbonic acid. This weak acid dissociates into a proton and a bicarbonate ion.
H+ are able to join the GIS radicals 14 6 in α- and β-chains of hemoglobin, i.e. in areas far from the heme. Protonation of hemoglobin reduces its affinity for O 2, promotes the elimination of O 2 from oxyHb, the formation of deoxyHb, and increases the supply of oxygen to tissues in proportion to the number of protons formed (Fig. 1.25).
The increase in the amount of released oxygen depending on the increase in the concentration of H + in erythrocytes is called the Bohr effect (after the Danish physiologist Christian Bohr, who first discovered this effect).
In the lungs, a high partial pressure of oxygen promotes its binding to deoxyHb, which reduces the protein's affinity for H+. The released protons under the action of carbanhydrase interact with bicarbonates to form CO 2 and H 2 O
Rice. 1.25. The dependence of the affinity of Hb to O 2 on the concentration of CO 2 and protons (Bohr effect):
A- influence of CO 2 and H+ concentration on the release of O 2 from the complex with Hb (Bohr effect); B- oxygenation of deoxyhemoglobin in the lungs, formation and release of CO 2 .
The resulting CO 2 enters the alveolar space and is removed with exhaled air. Thus, the amount of oxygen released by hemoglobin in tissues is regulated by the products of catabolism of organic substances: the more intense the breakdown of substances, for example, during physical exertion, the higher the concentration of CO 2 and H + and the more oxygen the tissues receive as a result of a decrease in the affinity of H to O 2.
8. Allosteric regulation of Hb affinity for O 2 by a ligand - 2,3-bisphosphoglycerate. In erythrocytes, the allosteric ligand of hemoglobin, 2,3-bisphosphoglycerate (2,3-BPG), is synthesized from the product of glucose oxidation - 1,3-bisphosphoglycerate. Under normal conditions, the concentration of 2,3-BPG is high and comparable to that of Hb. 2,3-BPG has a strong negative charge of -5.
Bisphosphoglycerate in tissue capillaries, by binding to deoxyhemoglobin, increases the oxygen output in tissues, reducing the affinity of Hb to O 2 .
There is a cavity in the center of the tetrameric hemoglobin molecule. It is formed by the amino acid residues of all four protomers (see Fig. 1.22). In tissue capillaries, the protonation of Hb (the Bohr effect) breaks the bond between the heme iron and O 2 . In a molecule
deoxyhemoglobin, compared with oxyhemoglobin, additional ionic bonds appear that connect the protomers, as a result of which the size of the central cavity increases compared to oxyhemoglobin. The central cavity is the site of attachment of 2,3-BPG to hemoglobin. Due to the difference in the size of the central cavity, 2,3-BPG can only attach to deoxyhemoglobin.
2,3-BPG interacts with hemoglobin in a region remote from active sites of the protein and belongs to allosteric(regulatory) ligands, and the central cavity Hb is allosteric center. 2,3-BPG has a strong negative charge and interacts with five positively charged groups of two Hb β-chains: the N-terminal α-amino group Val and the Lys 82 Gis 143 radicals (Fig. 1.26).
Rice. 1.26. BPG in the central cavity of deoxyhemoglobin
BPG binds to three positively charged groups in each β-strand.
In tissue capillaries, the resulting deoxyhemoglobin interacts with 2,3-BPG, and ionic bonds are formed between the positively charged radicals of β-chains and the negatively charged ligand, which change the protein conformation and reduce the affinity of Hb for O 2 . A decrease in the affinity of Hb for O 2 contributes to a more efficient release of O 2 into the tissue.
In the lungs, at high partial pressure, oxygen interacts with Hb, joining the heme iron; in this case, the conformation of the protein changes, the central cavity decreases, and 2,3-BPG is displaced from the allosteric center
Thus, oligomeric proteins have new properties compared to monomeric proteins. Attachment of ligands at sites,
spatially distant from each other (allosteric), capable of causing conformational changes in the entire protein molecule. Due to the interaction with regulatory ligands, the conformation changes and the function of the protein molecule adapts to environmental changes.
TOPIC 1.5. MAINTENANCE OF THE NATIVE CONFORMATION OF PROTEINS UNDER CELL CONDITIONS
In cells, during the synthesis of polypeptide chains, their transport through membranes to the corresponding sections of the cell, in the process of folding (formation of a native conformation) and during the assembly of oligomeric proteins, as well as during their functioning, intermediate, aggregation-prone, unstable conformations arise in the protein structure. Hydrophobic radicals, usually hidden inside the protein molecule in their native conformation, appear on the surface in an unstable conformation and tend to combine with groups of other proteins that are similarly poorly soluble in water. In the cells of all known organisms, special proteins have been found that provide optimal folding of cell proteins, stabilize their native conformation during functioning, and, most importantly, maintain the structure and functions of intracellular proteins in case of homeostasis disturbance. These proteins are called "chaperones" which means "nanny" in French.
1. Molecular chaperones and their role in preventing protein denaturation.
Chaperones (III) are classified according to the mass of subunits. High molecular weight chaperones have a mass of 60 to 110 kD. Among them, three classes have been studied the most: Sh-60, Sh-70 and Sh-90. Each class includes a family of related proteins. Thus, Sh-70 contains proteins with a molecular weight of 66 to 78 kD. Low molecular weight chaperones have a molecular weight of 40 to 15 kD.
Among the chaperones there are constitutive proteins whose high basal synthesis does not depend on stressful effects on the cells of the body, and inducible, the synthesis of which under normal conditions is weak, but increases sharply under stressful influences. Inducible chaperones are also called "heat shock proteins" because they were first discovered in cells exposed to high temperatures. In cells, due to the high concentration of proteins, spontaneous regeneration of partially denatured proteins is difficult. Sh-70 can prevent the process of denaturation that has begun and help restore the native conformation of proteins. Molecular chaperones-70- a highly conserved class of proteins found in all parts of the cell: cytoplasm, nucleus, endoplasmic reticulum, mitochondria. At the carboxyl end of the only polypeptide chain of Sh-70, there is a region that is a groove that can interact with peptides of length
7 to 9 amino acid residues enriched in hydrophobic radicals. Such sites in globular proteins occur approximately every 16 amino acids. Sh-70 are able to protect proteins from thermal inactivation and restore the conformation and activity of partially denatured proteins.
2. Role of chaperones in protein folding. During the synthesis of proteins on the ribosome, the N-terminal region of the polypeptide is synthesized before the C-terminal region. The complete amino acid sequence of the protein is required to form the native conformation. In the process of protein synthesis, chaperones-70, due to the structure of their active center, are able to cover polypeptide sites prone to aggregation enriched in hydrophobic amino acid radicals until synthesis is completed (Figure 1.27, A).
Rice. 1.27. Involvement of chaperones in protein folding
A - participation of chaperones-70 in the prevention of hydrophobic interactions between the sites of the synthesized polypeptide; B - formation of the native conformation of the protein in the chaperone complex
Many high molecular weight proteins with a complex conformation, such as a domain structure, fold in a special space formed by W-60. Sh-60 function as an oligomeric complex consisting of 14 subunits. They form two hollow rings, each of which consists of seven subunits, these rings are connected to each other. Each subunit of III-60 consists of three domains: apical (apical), enriched with hydrophobic radicals facing the cavity of the ring, intermediate and equatorial (Fig. 1.28).
Rice. 1.28. Structure of the chaperonin complex consisting of 14 Sh-60
A - side view; B - top view
Synthesized proteins with surface elements characteristic of unfolded molecules, in particular, hydrophobic radicals, enter the cavity of chaperone rings. In the specific environment of these cavities, an enumeration of possible conformations takes place until the only, energetically most favorable one is found (Fig. 1.27, B). The formation of conformations and release of the protein is accompanied by ATP hydrolysis in the equatorial region. Typically, such chaperone-dependent folding requires a significant amount of energy.
In addition to participating in the formation of the three-dimensional structure of proteins and the renativation of partially denatured proteins, chaperones are also required for such fundamental processes as the assembly of oligomeric proteins, recognition and transport of denatured proteins into lysosomes, transport of proteins across membranes, and participation in the regulation of the activity of protein complexes.
TOPIC 1.6. VARIETY OF PROTEINS. PROTEIN FAMILIES ON THE EXAMPLE OF IMMUNOGLOBULINS
1. Proteins play a decisive role in the life of individual cells and the entire multicellular organism, and their functions are surprisingly diverse. This is determined by the peculiarities of the primary structure and conformations of proteins, the unique structure of the active center, and the ability to bind specific ligands.
Only a very small part of all possible variants of peptide chains can adopt a stable spatial structure; majority
of them can take many conformations with approximately the same Gibbs energy, but with various properties. The primary structure of most known proteins, selected by biological evolution, provides exceptional stability of one of the conformations, which determines the features of the functioning of this protein.
2. Protein families. Within the same biological species, substitutions of amino acid residues can lead to the emergence of different proteins that perform related functions and have homologous amino acid sequences. Such related proteins have strikingly similar conformations: the number and arrangement of α-helices and/or β-structures, and most of the turns and folds of the polypeptide chains are similar or identical. Proteins with homologous regions of the polypeptide chain, similar conformation and related functions are isolated into protein families. Examples of protein families: serine proteinases, immunoglobulin family, myoglobin family.
Serine proteinases- a family of proteins that perform the function of proteolytic enzymes. These include digestive enzymes - chymotrypsin, trypsin, elastase and many blood coagulation factors. These proteins have 40% identical amino acids and a very similar conformation (Fig. 1.29).
Rice. 1.29. Spatial structures of elastase (A) and chymotrypsin (B)
Some amino acid substitutions have led to a change in the substrate specificity of these proteins and the emergence of functional diversity within the family.
3. Family of immunoglobulins. In the work of the immune system huge role play proteins of the immunoglobulin superfamily, which includes three families of proteins:
Antibodies (immunoglobulins);
T-lymphocyte receptors;
Proteins of the major histocompatibility complex - MHC 1st and 2nd classes (Major Histocompatibility Complex).
All these proteins have a domain structure, consist of homologous immune-like domains and perform similar functions: they interact with foreign structures, either dissolved in the blood, lymph or intercellular fluid (antibodies), or located on the surface of cells (own or foreign).
4. Antibodies- specific proteins produced by B-lymphocytes in response to the ingestion of a foreign structure called antigen.
Features of the structure of antibodies
The simplest antibody molecules consist of four polypeptide chains: two identical light chains - L, containing about 220 amino acids, and two identical heavy chains - H, consisting of 440-700 amino acids. All four chains in an antibody molecule are connected by many non-covalent bonds and four disulfide bonds (Fig. 1.30).
Light chains of antibodies consist of two domains: variable (VL), located in the N-terminal region of the polypeptide chain, and constant (CL), located at the C-terminus. Heavy chains typically have four domains: one variable (VH) at the N-terminus and three constants (CH1, CH2, CH3) (see Figure 1.30). Each immunoglobulin domain has a β-pleated superstructure in which two cysteine residues are linked by a disulfide bond.
Between the two constant domains CH1 and CH2 there is a region containing a large number of proline residues, which prevent the formation of the secondary structure and the interaction of neighboring H-chains in this segment. This hinge region gives the antibody molecule flexibility. Between the variable domains of the heavy and light chains are two identical antigen-binding sites (active sites for binding antigens), so such antibodies are often called bivalents. The binding of an antigen to an antibody does not involve the entire amino acid sequence of the variable regions of both chains, but only 20-30 amino acids located in the hypervariable regions of each chain. It is these areas that determine the unique ability of each type of antibody to interact with the corresponding complementary antigen.
Antibodies are one of the body's lines of defense against invading foreign organisms. Their functioning can be divided into two stages: the first stage is the recognition and binding of an antigen on the surface of foreign organisms, which is possible due to the presence of antigen-binding sites in the antibody structure; the second stage is the initiation of the process of inactivation and destruction of the antigen. The specificity of the second stage depends on the class of antibodies. There are five classes of heavy chains that differ from each other in the structure of constant domains: α, δ, ε, γ and μ, according to which five classes of immunoglobulins are distinguished: A, D, E, G and M.
Structural features of heavy chains give the hinge regions and C-terminal regions of heavy chains a conformation characteristic of each class. Once an antigen binds to an antibody, conformational changes in the constant domains determine the pathway for removal of the antigen.
Rice. 1. 30. Domain structure of IgG
Immunoglobulins M
Immunoglobulins M have two forms.
Monomeric form- 1st class of antibodies produced by the developing B-lymphocyte. Subsequently, many B cells switch to producing other classes of antibodies, but with the same antigen-binding site. IgM is incorporated into the membrane and acts as an antigen-recognizing receptor. The incorporation of IgM into the cell membrane is possible due to the presence of 25 hydrophobic amino acid residues in the tail portion of the region.
Secretory form of IgM contains five monomeric subunits linked to each other by disulfide bonds and an additional polypeptide J-chain (Fig. 1.31). Heavy chain monomers of this form do not contain a hydrophobic tail. The pentamer has 10 antigen-binding sites and is therefore effective in recognizing and removing the antigen that has entered the body for the first time. The secretory form of IgM is the main class of antibodies secreted into the blood during the primary immune response. Binding of IgM to an antigen changes the conformation of IgM and induces its binding to the first protein component of the complement system (the complement system is a set of proteins involved in the destruction of the antigen) and activation of this system. If the antigen is located on the surface of the microorganism, the complement system causes a violation of the integrity cell membrane and death of the bacterial cell.
Immunoglobulins G
In quantitative terms, this class of immunoglobulins predominates in the blood (75% of all Ig). IgG - monomers, the main class of antibodies secreted into the blood during the secondary immune response. After the interaction of IgG with surface antigens of microorganisms, the antigen-antibody complex is able to bind and activate proteins of the complement system or can interact with specific receptors on macrophages and neutrophils. interaction with phagocytes
Rice. 1.31. The structure of the secretory form of IgM
to the absorption of antigen-antibody complexes and their destruction in phagosomes of cells. IgG is the only class of antibodies that can cross the placental barrier and protect the fetus from infections in utero.
Immunoglobulins A
Main class of antibodies present in secretions (milk, saliva, respiratory and intestinal secretions). IgA is secreted mainly in a dimeric form, where the monomers are linked to each other through an additional J-chain (Fig. 1.32).
IgA do not interact with the complement system and phagocytic cells, but by binding to microorganisms, antibodies prevent them from attaching to epithelial cells and penetrating into the body.
Immunoglobulins E
Immunoglobulins E are represented by monomers in which heavy ε-chains contain, as well as μ-chains of immunoglobulins M, one variable and four constant domains. IgE after secretion bind with their own
Rice. 1.32. Structure of IgA
C-terminal regions with corresponding receptors on the surface of mast cells and basophils. As a result, they become receptors for antigens on the surface of these cells (Fig. 1.33).
Rice. 1.33. Interaction of IgE with antigen on the surface of the mast cell
After the antigen is attached to the corresponding antigen-binding IgE sites, the cells receive a signal to secrete biologically active substances (histamine, serotonin), which are largely responsible for the development of an inflammatory reaction and for the manifestation of such allergic reactions as asthma, urticaria, hay fever.
Immunoglobulins D
Immunoglobulins D are found in serum in very a small amount, they are monomers. Heavy δ chains have one variable and three constant domains. IgD act as receptors for B-lymphocytes, other functions are still unknown. The interaction of specific antigens with receptors on the surface of B-lymphocytes (IgD) leads to the transmission of these signals into the cell and the activation of mechanisms that ensure the reproduction of this clone of lymphocytes.
TOPIC 1.7. PHYSICO-CHEMICAL PROPERTIES OF PROTEINS AND METHODS FOR THEIR SEPARATION
1. Individual proteins differ in their physicochemical properties:
The shape of the molecules;
Molecular weight;
The total charge, the value of which depends on the ratio of anionic and cationic groups of amino acids;
The ratio of polar and non-polar amino acid radicals on the surface of molecules;
Degrees of resistance to various denaturing agents.
2. The solubility of proteins depends on the properties of the proteins listed above, as well as on the composition of the medium in which the protein dissolves (pH values, salt composition, temperature, the presence of other organic substances that can interact with the protein). The magnitude of the charge of protein molecules is one of the factors affecting their solubility. When the charge is lost at the isoelectric point, proteins more easily aggregate and precipitate. This is especially true for denatured proteins, which have hydrophobic amino acid radicals on the surface.
On the surface of the protein molecule, there are both positively and negatively charged amino acid radicals. The number of these groups, and hence the total charge of proteins, depends on the pH of the medium, i.e. the ratio of the concentration of H + - and OH - groups. In an acidic environment an increase in the concentration of H+ leads to the suppression of the dissociation of carboxyl groups -COO - + H+ > -COOH and a decrease in the negative charge of proteins. In an alkaline environment, the binding of excess OH - protons formed during the dissociation of amino groups -NH 3 + + OH - - NH 2 + H 2 O with the formation of water, leads to a decrease in the positive charge of proteins. The pH value at which a protein has a net charge of zero is called isoelectric point (IEP). In IET, the number of positively and negatively charged groups is the same, i.e. the protein is in an isoelectric state.
3. Separation of individual proteins. Features of the structure and functioning of the body depend on the set of proteins synthesized in it. The study of the structure and properties of proteins is impossible without their isolation from the cell and purification from other proteins and organic molecules. The stages of isolation and purification of individual proteins:
cell destruction of the studied tissue and obtaining a homogenate.
Separation of the homogenate into fractions centrifugation, obtaining a nuclear, mitochondrial, cytosolic or other fraction containing the desired protein.
Selective heat denaturation- short-term heating of the protein solution, in which part of the denatured protein impurities can be removed (in the event that the protein is relatively thermally stable).
Salting out. Different proteins precipitate at different concentrations of salt in solution. By gradually increasing the salt concentration, it is possible to obtain a number of individual fractions with a predominant content of the secreted protein in one of them. The most commonly used fractionation of proteins is ammonium sulfate. Proteins with the lowest solubility precipitate at low salt concentrations.
Gel filtration- a method of sieving molecules through swollen Sephadex granules (three-dimensional dextran polysaccharide chains with pores). The rate of passage of proteins through a column filled with Sephadex will depend on their molecular weight: the smaller the mass of protein molecules, the easier they penetrate into the granules and stay there longer, the larger the mass, the faster they elute from the column.
Ultracentrifugation- a method consisting in the fact that proteins in a centrifuge tube are placed in the rotor of an ultracentrifuge. When the rotor rotates, the sedimentation rate of proteins is proportional to their molecular weight: fractions of heavier proteins are located closer to the bottom of the tube, lighter ones are closer to the surface.
electrophoresis- a method based on differences in the speed of movement of proteins in an electric field. This value is proportional to the charge of proteins. Protein electrophoresis is carried out on paper (in this case, the rate of movement of proteins is proportional only to their charge) or in a polyacrylamide gel with a certain pore size (the rate of movement of proteins is proportional to their charge and molecular weight).
Ion exchange chromatography- a fractionation method based on the binding of ionized groups of proteins with oppositely charged groups of ion-exchange resins (insoluble polymeric materials). The binding strength of a protein to a resin is proportional to the charge of the protein. Proteins adsorbed on the ion-exchange polymer can be washed off with increasing concentrations of NaCl solutions; the lower the protein charge, the lower the concentration of NaCl will be required to wash away the protein associated with the ionic groups of the resin.
Affinity chromatography- the most specific method for isolating individual proteins. A ligand of a protein is covalently attached to an inert polymer. When a protein solution is passed through a column with a polymer, due to the complementary binding of the protein to the ligand, only the protein specific for this ligand is adsorbed on the column.
Dialysis- a method used to remove low molecular weight compounds from a solution of an isolated protein. The method is based on the inability of proteins to pass through a semipermeable membrane, unlike low molecular weight substances. It is used to purify proteins from low molecular weight impurities, for example, from salts after salting out.
ASSIGNMENTS FOR EXTRACURRICULUM WORK
1. Fill in the table. 1.4.
Table 1.4. Comparative analysis structures and functions of related proteins - myoglobin and hemoglobin
a) remember the structure of the active center Mb and Hb. What role do the hydrophobic radicals of amino acids play in the formation of the active centers of these proteins? Describe the structure of the Mb and Hb active center and the mechanisms of ligand attachment to it. What role do His F 8 and His E 7 residues play in the functioning of the Mv and Hv active site?
b) what new properties compared to monomeric myoglobin does a closely related oligomeric protein, hemoglobin, have? Explain the role of cooperative changes in the conformation of protomers in the hemoglobin molecule, the effect of CO 2 and proton concentrations on the affinity of hemoglobin to oxygen, and the role of 2,3-BPG in the allosteric regulation of Hb function.
2. Describe the characteristics of molecular chaperones, paying attention to the relationship between their structure and function.
3. What proteins are grouped into families? Using the example of the immunoglobulin family, determine the similar structural features and related functions of the proteins of this family.
4. Often, purified individual proteins are required for biochemical and medical applications. Explain on what physicochemical properties of proteins the methods used for their separation and purification are based.
TASKS FOR SELF-CONTROL
1. Choose the correct answers.
Functions of hemoglobin:
A. O 2 transport from lungs to tissues B. H + transport from tissues to lungs
B. Maintaining a constant blood pH D. Transport of CO2 from lungs to tissues
D. Transport of CO 2 from tissues to the lungs
2. Choose the correct answers. ligandα -Hb protomer is: A. Heme
B. Oxygen
B. CO D. 2,3-BPG
D. β-Protomer
3. Choose the correct answers.
Hemoglobin is different from myoglobin:
A. Has a quaternary structure
B. The secondary structure is represented only by α-helices
B. Refers to complex proteins
D. Interacts with an allosteric ligand D. Covalently bound to heme
4. Choose the correct answers.
The affinity of Hb for O 2 decreases:
A. When one O 2 molecule is attached B. When one O 2 molecule is eliminated
B. When interacting with 2,3-BPG
D. When attached to protomers H + D. When the concentration of 2,3-BPG decreases
5. Set a match.
For types Hb it is characteristic:
A. Forms fibrillar aggregates in deoxy form B. Contains two α- and two δ-chains
B. The predominant form of Hb in adult erythrocytes D. It contains heme with Fe + 3 in the active center
D. Contains two α- and two γ-chains 1. HvA 2.
6. Set a match.
Ligands Hb:
A. Binds to Hb at the allosteric center
B. Has a very high affinity for the active site Hb
B. Joining, increases the affinity of Hb to O 2 D. Oxidizes Fe + 2 to Fe + 3
D. Forms a covalent bond with hysF8
7. Choose the correct answers.
Chaperones:
A. Proteins present in all parts of the cell
B. Synthesis is enhanced under stressful influences
B. Participate in the hydrolysis of denatured proteins
D. Participate in maintaining the native conformation of proteins
D. Create organelles in which protein conformation is formed
8. Match. Immunoglobulins:
A. The secretory form is pentameric
B. Ig class that crosses the placental barrier
B. Ig - mast cell receptor
D. The main class of Ig present in the secretions of epithelial cells. D. B-lymphocyte receptor, the activation of which ensures cell reproduction
9. Choose the correct answers.
Immunoglobulins E:
A. Produced by macrophages B. Have heavy ε-chains.
B. Embedded in the membrane of T-lymphocytes
D. Act as membrane receptors for antigens on mast cells and basophils
D. Responsible for the manifestation of allergic reactions
10. Choose the correct answers.
The method for separating proteins is based on differences in their molecular weight:
A. Gel filtration
B. Ultracentrifugation
B. Polyacrylamide gel electrophoresis D. Ion exchange chromatography
D. Affinity chromatography
11. Choose the correct answer.
The method for separating proteins is based on differences in their solubility in water:
A. Gel filtration B. Salting out
B. Ion exchange chromatography D. Affinity chromatography
E. Polyacrylamide gel electrophoresis
STANDARDS OF ANSWERS TO "TASKS FOR SELF-CONTROL"
1. A, B, C, D
2. A, B, C, D
5. 1-B, 2-A, 3-D
6. 1-C, 2-B, 3-A
7. A, B, D, D
8. 1-G; 2-B, 3-C
BASIC TERMS AND CONCEPTS
1. Oligomeric proteins, protomer, quaternary structure of proteins
2. Cooperative changes in protomer conformation
3. Bohr effect
4. Allosteric regulation of protein functions, allosteric center and allosteric effector
5. Molecular chaperones, heat shock proteins
6. Protein families (serine proteases, immunoglobulins)
7. IgM-, G-, E-, A-connection of structure with function
8. Total charge of proteins, isoelectric point of proteins
9. Electrophoresis
10. Salting out
11. Gel filtration
12. Ion exchange chromatography
13. Ultracentrifugation
14. Affinity chromatography
15. Plasma protein electrophoresis
TASKS FOR AUDITIONAL WORK
1. Compare the dependences of the degrees of saturation of hemoglobin (Hb) and myoglobin (Mb) with oxygen on its partial pressure in tissues
Rice. 1.34. Saturation dependence of MV andHboxygen from its partial pressure
Please note that the shape of the protein oxygen saturation curves is different: for myoglobin - hyperbole, for hemoglobin - sigmoid shape.
1. Compare the values of the partial pressure of oxygen at which Mb and Hb are saturated with O 2 by 50%. Which of these proteins has a higher affinity for O 2 ?
2. What structural features of MB determine its high affinity for O 2 ?
3. What structural features of Hb allow it to release O 2 in the capillaries of resting tissues (at a relatively high partial pressure of O 2) and sharply increase this return in working muscles? What property of oligomeric proteins provides this effect?
4. Calculate what amount of O 2 (in%) gives oxygenated hemoglobin to the resting and working muscle?
5. draw conclusions about the relationship between protein structure and its function.
2. The amount of oxygen released by hemoglobin in capillaries depends on the intensity of catabolism processes in tissues (Bohr effect). How do changes in tissue metabolism regulate the affinity of Hb for O 2 ? Effect of CO 2 and H+ on the affinity of Hb for O 2
1. Describe the Bohr effect.
2. in what direction does the process shown in the diagram flow:
a) in the capillaries of the lungs;
b) in tissue capillaries?
3. What is the physiological significance of the Bohr effect?
4. Why does the interaction of Hb with H+ at sites remote from heme change the affinity of the protein for O 2 ?
3. The affinity of Hb to O 2 depends on the concentration of its ligand, 2,3-biphosphoglycerate, which is an allosteric regulator of the affinity of Hb to O 2 . Why does ligand interaction at a site remote from the active site affect protein function? How does 2,3-BPG regulate the affinity of Hb for O 2 ? To solve the problem, answer the following questions:
1. Where and from what is 2,3-biphosphoglycerate (2,3-BPG) synthesized? Write its formula, indicate the charge of this molecule.
2. What form of hemoglobin (oxy or deoxy) does BPG interact with and why? In which region of the Hb molecule does the interaction take place?
3. in what direction does the process shown in the diagram proceed?
a) in tissue capillaries;
b) in the capillaries of the lungs?
4. where should be the highest concentration of the complex
Nv-2,3-BFG:
a) in the capillaries of muscles at rest,
b) in the capillaries of working muscles (assuming the same concentration of BPG in erythrocytes)?
5. How will the affinity of Hb for oxygen change when a person adapts to high altitude conditions, if the concentration of BPG in erythrocytes increases? What is the physiological significance of this phenomenon?
4. The destruction of 2,3-BPG during storage of preserved blood disrupts the functions of Hb. How will the affinity of Hb to O 2 in preserved blood change if the concentration of 2,3-BPG in erythrocytes can decrease from 8 to 0.5 mmol/l. Is it possible to transfuse such blood to seriously ill patients if the concentration of 2,3-BPG is restored no earlier than after three days? Is it possible to restore the functions of erythrocytes by adding 2,3-BPG to the blood?
5. Recall the structure of the simplest immunoglobulin molecules. What role do immunoglobulins play in the immune system? Why are Igs often referred to as bivalents? How is the structure of Igs related to their function? (Describe using an example of a class of immunoglobulins.)
Physico-chemical properties of proteins and methods for their separation.
6. How does the net charge of a protein affect its solubility?
a) determine the total charge of the peptide at pH 7
Ala-Glu-Tre-Pro-Asp-Liz-Cis
b) how will the charge of this peptide change at pH >7, pH<7, рН <<7?
c) what is the isoelectric point of a protein (IEP) and in what environment does it lie
IET of this peptide?
d) at what pH value will the least solubility of this peptide be observed.
7. Why does sour milk, unlike fresh milk, “coagulate” when boiled (i.e., casein milk protein precipitates)? Casein molecules in fresh milk have a negative charge.
8. Gel filtration is used to separate individual proteins. A mixture containing proteins A, B, C with molecular masses equal to 160,000, 80,000 and 60,000, respectively, was analyzed by gel filtration (Fig. 1.35). Swollen gel granules are permeable to proteins with a molecular weight of less than 70,000. What principle underlies this separation method? Which of the graphs correctly represents the results of fractionation? Specify the order of release of proteins A, B and C from the column.
Rice. 1.35. Using the Gel Filtration Method to Separate Proteins
9. On fig. 1.36, A shows a diagram of electrophoresis on paper of proteins in the blood serum of a healthy person. The relative amounts of protein fractions obtained using this method are: albumins 54-58%, α 1 -globulins 6-7%, α 2 -globulins 8-9%, β-globulins 13%, γ-globulins 11-12% .
Rice. 1.36 Electrophoresis on paper of blood plasma proteins of a healthy person (A) and a patient (B)
I - γ-globulins; II - β-globulins; III -α 2 - globulin; IV-α 2 - globulin; V - albumins
Many diseases are accompanied by quantitative changes in the composition of whey proteins (dysproteinemia). The nature of these changes is taken into account when making a diagnosis and assessing the severity and stage of the disease.
Using the data given in table. 1.5, make an assumption about the disease, which is characterized by the electrophoretic profile presented in fig. 1.36.
Table 1.5. Changes in the concentration of blood serum proteins in pathology