BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E. S. Severin - 2004

SECTION 1. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

II. Protein Structure

Peptide chains contain tens, hundreds, and thousands of amino acid residues linked by strong peptide bonds. Through intramolecular interactions, Proteins form a specific spatial architecture known as "protein conformation." The linear sequence of Amino Acids within a protein contains the blueprint for constructing its three-dimensional Spatial Structure. There are four hierarchical Levels of Protein structural Organization, referred to as primary, secondary, tertiary, and quaternary structures (Figs. 1–3). Universal principles govern the folding and formation of these spatial protein structures.

Class="center">Fig. 1-3. Stages of Protein conformation formation. 1 — Primary Structure; 2 — Secondary structure; 3 — tertiary structure; 4 — quaternary structure.

A. Primary Structure

Amino acid residues in a protein's peptide chain do not alternate at random; rather, they are arranged in a precise, defined sequence. The linear sequence of amino acid residues in a polypeptide chain is called the "Primary Cell/13.html">Protein Structure."

The Introduction/19.html">Primary structure of each individual protein is encoded within a specific segment of DNA known as a Gene. During Protein Synthesis, the Genetic information is first transcribed into mRNA, and then, using the mRNA as a template, the primary structure is assembled on the ribosome (see Section 4).

Each of the 50,000 individual proteins in The Human Body possesses a unique primary structure specific to that protein. All molecules of a given individual protein share the exact same sequence of amino acid residues, which is the primary feature distinguishing it from any other protein.

B. Methods FOR STUDYING Protein Primary Structure

Investigating the Primary Structure of Proteins is of paramount general biological and medical importance. By examining The sequence of amino acid residues in individual proteins and correlating this data with the spatial arrangement of the molecule, researchers can uncover fundamental universal patterns governing protein folding.

Furthermore, many Genetic Disorders result from abnormalities in protein Amino acid sequences. Information regarding the primary structure of both normal and mutant Proteins can be invaluable for diagnosing and prognosticating the course of diseases.

Determining the primary structure of proteins involves two main stages:

✵ determining the Amino Acid Composition of the protein under study;

✵ determining the exact Amino Acid Sequence within the protein.

1. Determination of protein Amino Acid Composition

The first step in elucidating a protein's primary structure involves a Qualitative and quantitative assessment of its amino acid composition. It is essential to note that this analysis requires a specific quantity of purified protein, free from contaminants such as other proteins or Peptides.

Acid Hydrolysis of Proteins

To determine The amino acid composition, all peptide bonds in the protein must be completely cleaved. The protein sample is hydrolyzed in 6 mol/L HCl at approximately 110 °C for 24 hours. This Treatment breaks all peptide bonds, leaving only free amino acids in the hydrolyzate. In addition, glutamine and asparagine are hydrolyzed to glutamic and aspartic acids, respectively (i.e., the amide bond in their side chains is cleaved, releasing the amino group).

Separation of Amino Acids via Ion-exchange Chromatography

The mixture of amino acids obtained from acid hydrolysis is separated using a Column packed with a cation-exchange resin. Such synthetic resins contain firmly bound negatively charged groups (e.g., sulfonic acid residues -SO₃^-) associated with Na⁺ ions (Fig. 1–4).

Fig. 1-4. Separation of amino acids by ion-exchange chromatography. A. Chromatographic column packed with cation-exchange resin. B. Stages of amino acid separation: 1 — binding of amino acids to resin particles; 2 — elution of amino acids at specific pH values and NaCl concentrations.

The amino acid mixture is loaded onto the cation exchanger in an acidic medium (pH 3.0), where the amino acids predominantly exist as cations, carrying a positive net charge. These positively charged amino acids bind to the negatively charged resin particles. The greater the net positive charge of an amino acid, the stronger its binding affinity to the resin. Consequently, the amino acids Lysine, Arginine, and Histidine bind most tightly to the cation exchanger, whereas aspartic and glutamic acids bind the least tightly.

Amino acids are eluted from the column using a buffer solution with an increasing Ionic strength (i.e., an increasing NaCl concentration) and pH. As the pH rises, amino acids lose a proton, which reduces their positive charge and, consequently, the strength of their binding to the negatively charged resin particles.

Each amino acid emerges from the column at a specific pH and ionic strength. By collecting the solution (eluate) from the bottom of the column in small portions, fractions containing individual Amino acids can be obtained.

Quantitative Analysis of the obtained fractions

The amount of each amino acid in a given protein is determined by heating individual amino acid fractions with ninhydrin, which forms a red-purple compound. The color intensity in the sample is proportional to the amount of the amino acid present; therefore, by spectrophotometrically measuring the light absorbed by the ninhydrin derivatives, the content of each amino acid in the protein hydrolysate can be determined.

Currently, The process of separating and quantifying amino acids in a protein hydrolysate is fully automated and performed using a specialized instrument known as an amino acid analyzer.

2. Determination of the amino acid sequence in a protein

Determination of the N-terminal amino acid in a protein and the amino acid sequence in oligopeptides

Phenylisothiocyanate (PITC) is a reagent used to determine the N-terminal amino acid in a peptide. It is capable of reacting with the α-amino and α-carboxyl groups of free amino acids, as well as with the N-terminal amino acid in peptides (see the scheme below).

The reaction with the N-terminal amino acid of a polypeptide yields a phenylthiohydantoin derivative, in which the peptide bond between the α-carboxyl group of the N-terminal Amino Acid and the α-amino group of the second amino acid in the peptide is destabilized. This bond is selectively hydrolyzed without damaging other peptide bonds.

Following the reaction, the PITC-AA1 complex is isolated and identified using chromatographic methods. PITC can be reused with the shortened peptide obtained in the previous cycle to identify the next amino acid. This stepwise peptide degradation process from the N-terminus has been automated and implemented in an instrument called a Sequencer, which makes it possible to determine the amino acid sequence of oligopeptides consisting of 10 to 20 amino acids.

Many Polypeptides have a primary structure comprising more than 100 amino acids. Because sequencers are most efficient at determining the amino acid sequence of only relatively small peptides, polypeptide molecules are cleaved into fragments at specific sites.

By using several different Cleavage agents (which can be Enzymes or chemical Reagents) on separate samples of the purified polypeptide, partially overlapping fragments with established amino acid sequences can be obtained. These allow the correct order of the fragments to be reconstructed and the complete amino acid sequence of the polypeptide chain to be determined.

Enzymatic cleavage of a polypeptide at specific sites

Several different Enzymes can be used for the Specific Cleavage of peptide bonds in a protein. The most widely used method is the Enzymatic hydrolysis of a polypeptide by the proteolytic enzyme Trypsin, which belongs to the group of digestive enzymes produced by the Pancreas. The enzyme exhibits high Specificity, cleaving peptide bonds formed by the carboxyl group of lysine or arginine residues.

Based on the established number of lysine and arginine residues, the number of fragments produced by trypsin hydrolysis can be predicted. For instance, if a polypeptide chain contains 6 arginine and lysine residues, tryptic Digestion will yield 7 fragments.

Scheme

The amino acid sequence of each fragment is then determined.

Chemical cleavage of a polypeptide at specific sites

In some cases, chemical hydrolysis is preferred over enzymatic cleavage. For example, the reagent Cyanogen bromide exclusively cleaves peptide bonds where the carboxyl group belongs to a Methionine residue. Knowing the number of methionine residues in the polypeptide chain makes it easy to calculate the number of fragments produced. The amino acid sequence of each fragment is subsequently determined using a sequencer.

Determination of the polypeptide amino acid sequence using overlapping fragments

To successfully sequence the obtained polypeptide fragments, it is necessary to generate peptides with overlapping amino acid sequences. This is achieved by treating separate samples of the given polypeptide with different reagents that cleave the protein at different sites. A sufficient number of cleavages must be performed to yield a set of peptides that provide overlapping coverage across all regions required to determine the sequence of the original polypeptide.

Determination of protein primary structure using overlapping peptide fragments.

B. Protein Conformation

Linear polypeptide chains of individual proteins acquire a specific three-dimensional spatial structure, known as conformation, through the interactions of amino acid functional groups. All molecules of a given individual protein (i.e., those with identical primary structure) adopt the exact same conformation in solution. Consequently, all the information required to form these spatial structures is inherently contained within the primary STRUCTURE OF THE proteins.

Proteins generally exhibit two MAIN TYPES OF polypeptide chain conformation: secondary and tertiary structures.

1. Protein secondary structure

Protein secondary structure is the spatial arrangement formed by interactions between functional groups within the peptide backbone. Through these interactions, peptide chains can adopt two regular structural types: the α-Helix and the β-structure.

α-Helix

In this type of structure, the peptide backbone coils into a helix due to The formation of Hydrogen Bonds between the carbonyl oxygen atoms and the amino nitrogen atoms of peptide groups located four amino acid residues apart. These Hydrogen bonds are oriented parallel to the axis of the helix (Figs. 1–5). Each turn of the α-helix comprises 3.6 amino acid residues.

Fig. 1-5. The α-helix. The diagram illustrates the spatial architecture of an α-helical segment of a polypeptide chain and the hydrogen bonds involved in forming the α-helix.

Virtually all oxygen and hydrogen atoms of the peptide groups participate in hydrogen bonding. As a result, the α-helix is tightly stabilized by a multitude of hydrogen bonds. Although these bonds are individually weak, their sheer number ensures the maximum possible Stability of the α-helix. Because all hydrophilic groups of the peptide backbone typically engage in hydrogen bonding, the hydrophilicity (i.e., The ability to form hydrogen bonds with Water) of α-helices decreases, while their Hydrophobicity increases.

The α-helical structure represents the most stable conformation of the peptide backbone, corresponding to a state of minimum Free energy. The formation of α-helices shortens the polypeptide chain, but if conditions are introduced that disrupt these hydrogen bonds, the chain will elongate once again.

Amino acid side chains (radicals) are positioned on the outer surface of the α-helix, projecting outward from the peptide backbone. They do not take part in the hydrogen bonds characteristic of secondary structure, though certain residues can disrupt α-helix formation. These include:

✵ Proline: its nitrogen atom is part of a rigid ring structure, which precludes rotation around the -N-CH- bond. Furthermore, the proline nitrogen atom involved in the peptide bond with another amino acid lacks a hydrogen atom. Consequently, proline cannot form a Hydrogen bond at that specific position in the peptide backbone, thereby disrupting the α-helix and typically introducing a loop or a bend into the chain;

✵ segments where several similarly charged side chains occur consecutively, generating electrostatic repulsive forces between them;

✵ regions containing bulky side chains located in close proximity that mechanically hinder α-helix formation, such as methionine or Tryptophan.

β-Structure

The β-structure is formed by extensive hydrogen bonding between the peptide groups of linear regions within a single folding polypeptide chain, or between different polypeptide chains. The β-structure forms an accordion-like, pleated sheet, known as the β-pleated sheet (Fig. 1-6).

Fig. 1-6. Protein secondary structure in the form of a β-pleated sheet.

When hydrogen bonds form between the peptide backbone atoms of separate polypeptide chains, they are referred to as intermolecular bonds. Hydrogen bonds occurring between linear segments within a single polypeptide chain are called intramolecular bonds. In β-structures, these hydrogen bonds are oriented perpendicular to the polypeptide chain.

If the bonded polypeptide chains run in opposite directions, an antiparallel β-structure is formed; if the N- and C-termini of the chains are aligned in the same direction, a parallel β-pleated sheet structure is produced (Fig. 1-7).

Fig. 1-7. Parallel and antiparallel β-pleated sheets. β-Structures are indicated by broad arrows. A - antiparallel β-structure; B - parallel β-pleated sheet structures.

Unlike α-helices, the disruption of hydrogen bonds forming β-structures does not lead to the elongation of those polypeptide chain segments.

Both the α-helix and β-pleated sheets are found in both globular and Fibrous proteins.

Irregular Secondary Structures

Proteins also feature regions with irregular secondary structures, commonly referred to as random coils. These consist of loop- and ring-like Conformations that exhibit lower structural regularity than the α-helices and β-sheets described above. Nevertheless, they do not vary wildly from one protein molecule to another. In each individual protein, they maintain a fixed conformation determined by the amino acid composition of that specific segment and its neighboring regions.

The term "random coil" is also frequently used to describe a denatured protein that has lost its ordered structure following the disruption of weak intramolecular bonds.

Distribution of Different Secondary Structure Types in Proteins

The proportion of the aforementioned secondary structures varies significantly among different proteins. Based on the presence of α-helices and β-sheets, Globular proteins can be divided into four categories.

✵ The first category comprises proteins whose structures consist exclusively of α-helices. Examples include proteins such as Myoglobin and Hemoglobin (Fig. 1-8).

Fig. 1-8. Eight α-helices in The structure of myoglobin (A) and β-chains of hemoglobin (B).

✵ The second category includes proteins containing both α-helices and β-sheets, which sometimes form characteristic combinations shared across different individual proteins (Fig. 1-9). Typical combinations of α-helices and β-sheets found in many enzymes can be illustrated by the domain structures of Lactate dehydrogenase (LDH) and phosphoglycerate kinase (PGK). A domain is a region of a polypeptide chain that independently folds into a structure largely resembling a globular protein.

Fig. 1-9. α-Helices and β-sheets in the domain of lactate dehydrogenase (A) and phosphoglycerate kinase (B).

In one of the domains of lactate dehydrogenase, the center is occupied by β-sheets of the polypeptide chain arranged in a twisted sheet, with each β-sheet connected to an α-helical segment located on the molecular surface. As seen in Fig. 1-9, a similar domain is also present in the phosphoglycerate kinase molecule.

✵ The third category encompasses proteins that possess only β-sheets. Such structures are found in IMMUNOGLOBULINS and the enzyme superoxide dismutase (Fig. 1-10).

Fig. 1-10. β-Pleated secondary structure in the constant domain of an immunoglobulin (A) and the enzyme superoxide dismutase (B).

✵ The fourth category includes proteins that contain only a negligible amount of regular secondary structures.

2. Tertiary Structure of Proteins

Protein tertiary structure — the three-dimensional spatial conformation formed through interactions between amino acid side chains (radicals) that may be located at considerable distances from one another along the polypeptide chain.

Bonds Involved in the Formation of Protein Tertiary Structure

Hydrophobic Interactions

During folding, a protein's polypeptide chain tends to adopt an energetically favorable conformation characterized by a minimum of free energy. Consequently, hydrophobic amino acid side chains tend to cluster together within the interior of water-soluble globular proteins. This gives rise to so-called hydrophobic interactions, as well as Van der Waals forces between closely packed atoms. As a result, a Hydrophobic core is formed inside the protein globule. The hydrophilic groups of the peptide backbone form numerous hydrogen bonds during secondary structure formation, thereby preventing water molecules from binding to them and disrupting the dense internal protein structure.

Ionic and Hydrogen Bonds

Hydrophilic amino acid side chains tend to form hydrogen bonds with water and are therefore predominantly located On the surface of the protein molecule.

All hydrophilic side chain groups trapped within the hydrophobic core interact with one another via ionic and hydrogen bonds (Fig. 1-11).

Fig. 1-11. Types of bonds formed between amino acid side chains during the folding of the protein tertiary structure. 1 — ionic bonds; 2 — hydrogen bonds; 3 — Hydrophobic bonds; 4 — Disulfide Bonds.

Ionic bonds can form between negatively charged (anionic) carboxyl groups of aspartic and glutamic acid side chains and positively charged (cationic) groups of lysine, arginine, or histidine side chains.

Hydrogen bonds form between hydrophilic uncharged groups (such as -OH, -СОNН₂, and SН groups) and any other hydrophilic groups.

Proteins functioning in a non-polar (lipid) environment, such as Membrane Proteins, exhibit an inverse arrangement: hydrophilic amino acid side chains are located inside the protein, whereas hydrophobic amino acids are localized on the molecular surface and interact with the non-polar environment. In each case, amino acid side chains adopt the most energetically favorable position.

Covalent bonds

The tertiary structure of certain proteins is stabilized by disulfide bonds, which form through the interaction of SН groups from two Cysteine residues. Although these two cysteine residues may be located far apart in the linear primary structure of the protein, they are brought into close proximity during tertiary folding to form a stable covalent cross-link between the side chains (Fig. 1-12).

Fig. 1-12. Formation of a disulfide bond in proteins.

Most intracellular proteins lack disulfide bonds. However, such bonds are widespread in proteins secreted by Cells into the extracellular space. These covalent bonds are believed to stabilize protein conformation outside The Cell and prevent Denaturation. Examples of such proteins include the hormone Insulin and immunoglobulins.

Insulin is a protein hormone containing 51 Amino Acids and consisting of two polypeptide chains (chain A contains 21 amino acids, and chain B contains 30 amino acids). Insulin is synthesized in the pancreatic β-cells and secreted into the Blood in response to elevated blood glucose levels. The structure of insulin features 2 disulfide bonds connecting the A and B polypeptide chains, and 1 intrachain disulfide bond within chain A (Fig. 1-13). The structure of immunoglobulins is discussed in subsection 6 D.

Fig. 1-13. Disulfide bonds in the structure of the insulin hormone.

All proteins with the identical primary structure under the same conditions acquire the same conformation, characteristic of that specific individual protein, which determines its specific function. The functionally active conformation of a protein is referred to as the “native structure.”

3. Conformational flexibility of proteins

Hydrophobic interactions, as well as ionic and hydrogen bonds, are classified as weak bonds because their energy only slightly exceeds the thermal motion energy of atoms at room Temperature (i.e., these bonds can already be disrupted at this temperature). Maintaining a protein's characteristic conformation is made possible by the formation of numerous weak bonds between different Regions of the polypeptide chain.

However, proteins consist of a vast number of atoms undergoing constant (Brownian) motion, which leads to slight displacements of individual polypeptide segments that typically do not disrupt the overall protein Structure and function. Consequently, proteins exhibit conformational flexibility — a propensity for minor conformational shifts resulting from the breaking of some weak bonds and the formation of others. A protein's conformation can change in response to alterations in the chemical and Physical Properties of the environment, as well as upon interaction with other molecules. This alters the spatial structure not only of the region contacting another molecule but also of the protein conformation as a whole. Conformational changes play a crucial role in the functioning of proteins within a living cell.

4. Protein Denaturation

The disruption of A large number of weak bonds in a protein molecule leads to The breakdown of its native conformation. Because bond cleavage under METABOLISM/18.html">The Influence of various factors occurs randomly, molecules of an individual protein in solution acquire the shape of randomly formed, disordered coils that differ from one another in their three-dimensional structure. The loss of the native conformation is accompanied by the loss of specific Protein Functions. This process is known as protein denaturation. Protein denaturation does not involve the cleavage of peptide bonds, meaning the primary structure of the protein remains intact.

In a denatured protein, hydrophobic side chains that are hidden inside the hydrophobic core in the native structure end up on the surface. At a sufficiently high protein concentration and in the absence of a strong repulsive charge, molecules can aggregate through hydrophobic interactions, leading to a decrease in Protein solubility and precipitation.

The compact, dense Spatial structure of a native protein dramatically expands upon denaturation, making it readily accessible to cleavage of peptide bonds by Proteolytic Enzymes (Fig. 1-14). Heat treatment of meat prior to consumption not only improves its palatability but also facilitates its Enzymatic Digestion in the digestive tract. Furthermore, the acidic environment of The Stomach exerts a denaturing effect on dietary proteins, causing the denaturation of those that have not undergone prior heat treatment, while also denaturing the proteins of microorganisms entering the stomach with food.

Fig. 1-14. Structure of a native protein molecule (center) and three denatured molecules of the same protein.

5. Factors causing protein denaturation

Protein denaturation is caused by factors that promote the disruption of hydrophobic, hydrogen, and ionic bonds that stabilize protein conformation:

✵ high temperatures (above 50 °C), which increase the thermal motion of atoms within the molecule and lead to the disruption of weak bonds;

✵ intense shaking of the solution, causing protein molecules to come into contact with the air at the phase boundary and altering their conformation;

✵ organic substances (such as ethanol, phenol, and its derivatives) capable of interacting with Functional groups of proteins, leading to conformational changes. In biochemical research, urea or guanidine hydrochloride are frequently used to denature proteins; these compounds form hydrogen bonds with the amino and carbonyl groups of the peptide backbone, as well as with certain functional groups of amino acid side chains. This breaks the bonds involved in forming the secondary and tertiary structure of native proteins and creates new bonds with chemical reagents;

✵ acids and alkalis, by altering the pH of the medium, cause a redistribution of bonds within the protein molecule;

✵ heavy metal salts (such as copper, mercury, silver, lead, etc.) form stable bonds with essential functional groups of proteins (most commonly -SH), altering their conformation and activity;

✵ detergents — substances containing a hydrophobic hydrocarbon radical and a hydrophilic functional group (such compounds are termed amphiphilic). The hydrophobic radicals of proteins interact with the hydrophobic parts of detergents, altering protein conformation. A protein denatured by detergents typically remains in a dissolved state, as the hydrophilic PARTS OF THE denaturing agent keep it in solution. Soaps are among the best-known examples of detergents (Fig. 1-15).

Fig. 1-15. Protein denaturation by detergents.

6. Medical aspects of conformational lability of proteins

The tendency of most proteins to denature during isolation, storage, and handling presents a major challenge in their production and medical application.

To ensure proper handling, protein-based Pharmaceuticals are supplied with instructions outlining their storage and usage conditions. For instance, most protein drugs must be stored in a refrigerator at temperatures not exceeding 10 °C, and dry preparations should be dissolved in boiled water cooled to room temperature to prevent denaturation.

7. Application of Denaturing Agents in biological research and medicine

In biochemical studies, low-molecular-weight compounds are typically isolated from biological samples after first removing proteins from the solution. Trichloroacetic acid is most commonly used for this purpose. Upon its addition, denatured proteins precipitate and are easily removed by filtration. Trichloroacetic acid can also be used to denature enzymes in order to halt enzymatic reactions.

In medicine, denaturing agents are frequently used for the sterilization of medical instruments and Materials, as well as serving as antiseptics. For example, medical instruments and supplies are sterilized in autoclaves at high temperatures.

Phenol and its derivatives (cresol, resorcinol) are well-known aromatic antiseptics. Due to their high hydrophobicity, they effectively target vegetative forms of Bacteria and Fungi by denaturing their proteins. The antimicrobial efficacy of these agents decreases as their water solubility increases.

A solution of cresol in potassium soap is known as lysol, which is used as a disinfectant.

Birch tar, one of the Main Components of Vishnevsky ointment, contains phenol. This preparation, used for wound healing, exhibits strong antimicrobial activity.

A significant number of antiseptics are heavy metal salts. Their antimicrobial action is due to the fact that, even at relatively low concentrations, they interact with microbial proteins, block their -SH groups, and alter their conformation. Because of their high toxicity, most medications containing heavy metal salts are used as topical antiseptics.

For instance, sublimate—mercury dichloride (HgCl₂)—possesses high antimicrobial activity. It is used for hand antisepsis and surface disinfection. Accidental or intentional poisoning with mercury preparations causes severe necrotic lesions of the digestive tract mucosa and necrotic Changes in the Kidneys. Silver preparations, such as silver nitrate (AgNO₃) and collargol (colloidal silver), also exhibit antimicrobial properties and are used to treat mucous membranes during infectious diseases.

G. Supersecondary structure of proteins

The spatial structure of each protein is unique and determined by its primary structure. However, comparing the conformations of proteins with different structures and functions has revealed the presence of similar combinations of secondary structure elements. This specific arrangement of secondary structures is referred to as the supersecondary structure of proteins. Supersecondary structure is formed through interactions between amino acid side chains.

Certain characteristic combinations of α-helices and β-structures are frequently designated as "Structural motifs." They have specific names: "α-helix — turn — α-helix," "β-barrel structure," "leucine zipper," "zinc finger," and others. The specific spatial arrangement of α-helices and β-structures is formed through interactions between side chains.

1. β-Barrel type supersecondary structure

This structure indeed resembles a barrel, where each β-structure (indicated by an arrow in Fig. 1-16) is located internally and connected to an α-helical region of the polypeptide chain situated on the protein surface.

Certain enzymes, such as Triosephosphate isomerase and one of the Pyruvate kinase domains (Fig. 1-16), exhibit a supersecondary structure in the form of a β-barrel.

Fig. 1-16. Supersecondary structure of a β-barrel. A - triosephosphate isomerase; B - pyruvate kinase domain.

2. The "Helix-Turn-Helix" Structural Motif

This structural motif is found in numerous DNA-binding proteins. The double-helical structure of DNA features two grooves: a major groove and a minor groove. The major groove is well-suited for binding proteins that contain small helical segments.

This structural motif comprises two α-helices—one shorter and one longer—connected by a turn of the polypeptide chain. The shorter α-helix lies across the groove, while the longer α-helix sits within the major groove, forming non-covalent specific interactions between amino acid side chains and DNA NUCLEOTIDES (Fig. 1-17).

Fig. 1-17. Binding of the "helix-turn-helix" supersecondary structure of a DNA-binding protein within the major groove of DNA.

3. Zinc Finger Supersecondary Structure

This type of supersecondary structure is also frequently observed in DNA-binding proteins. A zinc finger is a protein fragment containing approximately 20 amino acid residues, in which a zinc atom is coordinated to the side chains of four amino acids—typically two cysteine and two histidine residues. In some cases, cysteine residues also replace the histidines (Fig. 1-18).

Fig. 1-18. A zinc finger fragment of a DNA-binding protein.

Two closely spaced cysteine residues are separated from the other two residues (histidine or cysteine) by an amino acid sequence consisting of approximately 12 residues. This protein segment forms an α-helix that can specifically bind to regulatory regions in the major groove of DNA. The Specificity of the interaction between the DNA-binding protein and a specific DNA region depends on the amino acid sequence within the zinc finger domain.

4. Leucine Zipper Supersecondary Structure

Some DNA-binding proteins are oligomeric, meaning they consist of multiple polypeptide chains. Furthermore, certain proteins function in complexes with other proteins. The assembly of protomers or individual proteins into complexes is sometimes mediated by structural motifs known as leucine zippers.

On The surface of each interacting polypeptide chain or protein, there is an α-helical region containing at least 4 leucine residues. These leucine residues are spaced every 6 amino acids. Since each turn of an α-helix contains 3.6 amino acid residues, the leucine side chains appear on the surface of every second turn.

The leucine residues in the α-helix of one protein can interact with those of another protein via hydrophobic interactions, holding them together (Fig. 1-19).

Fig. 1-19. A "leucine zipper" between the α-helical regions of two proteins.

Histones serve as a classic example of protein association via a leucine zipper. Histones are Nuclear Proteins rich in positively charged amino acids, namely arginine and lysine. Histone molecules assemble into complexes consisting of 8 monomeric proteins via leucine zippers, despite the strong positive charge carried by all monomers.

D. Protein Domain Structure

If a protein polypeptide chain contains more than 200 amino acids, its spatial structure is typically organized into two or more domains. A domain is a region of a polypeptide chain that, during folding, acquires the conformation of a globular protein independently of other regions in the same chain. For instance, the light chain of immunoglobulin G consists of two domains. In some contexts, distinct structural segments of a polypeptide chain are also referred to as domains.

Domains can usually be isolated by treating the protein with proteolytic enzymes, which readily cleave peptide bonds in the polypeptide linker region situated between the domains. Following this cleavage, individual domains may retain their biological properties.

E. Quaternary Structure of Proteins

Many proteins consist of a single polypeptide chain. Such proteins are referred to as monomers. Monomeric proteins also include those composed of several chains covalently linked by, for example, disulfide bonds (hence insulin should be considered a monomeric protein).

At the same time, some proteins consist of two or more polypeptide chains. Once the three-dimensional structure of each polypeptide chain is formed, they associate via the same weak interactions that drive the formation of tertiary structure: hydrophobic, ionic, and hydrogen bonds.

The number and spatial arrangement of polypeptide chains are referred to as the quaternary structure of proteins. The individual polypeptide chains in such proteins are called protomers, or subunits. A protein containing multiple protomers is termed oligomeric.

1. Number of protomers in the structure of Oligomeric Proteins

Oligomeric proteins may contain anywhere from two to several dozen protomers, although the most common are proteins with two to four polypeptide chains (dimeric and tetrameric proteins).

For instance, the enzyme hexokinase contains 2 protomers; the erythrocyte protein hemoglobin and the enzyme lactate dehydrogenase contain 4 protomers; cytochrome c oxidase, an inner mitochondrial membrane enzyme, contains 13 protomers; and Glutamine Synthetase contains 12 protomers (Fig. 1-20). There are also large multifunctional complexes comprising several dozen polypeptide chains; for example, the pyruvate dehydrogenase complex consists of 312 protomers.

Fig. 1-20. Subunit structure of glutamine synthetase.

Some oligomeric proteins contain identical protomers (e.g., hexokinase), whereas others consist of different protomers. For example, hemoglobin contains 2 α- and 2 β-protomers, while lactate dehydrogenase, which has 4 protomers, features 2 types of monomers (H and M) that can occur in various combinations across different Tissues (e.g., 4H or 3H + 1M, etc.).

Oligomeric proteins have a high molecular weight. Proteins with a molecular weight exceeding 50,000 D almost invariably contain multiple monomeric polypeptide chains. Compared to individual monomeric proteins, oligomers perform more complex functions.

2. Assembly of protomers into an oligomeric protein. Protomers complementarity

The recognition and joining of individual protomers in an oligomeric protein occur through the formation of contact sites on their surfaces. These sites consist of amino acid side chains brought together in specific locations during the folding of the tertiary structure. Together, these side chains form unique surfaces capable of associating with one another with high specificity.

The binding specificity of contact sites is determined by their complementarity. Complementarity is the spatial and chemical correspondence between interacting surfaces. The depressions and protrusions on the surface of one molecule must match the protrusions and depressions on the surface of another, much like two pieces of torn paper. Additionally, functional groups of amino acid side chains on one contact surface must form weak chemical bonds with the amino acid side chains on the other surface (Fig. 1-21). Contact regions are typically rich in hydrophobic amino acid side chains, whose association forms the hydrophobic core of the oligomeric protein. Hydrophilic side chains can form hydrogen and ionic bonds.

Fig. 1-21. Schematic representation of dimeric protein molecule formation. Numerous weak bonds, indicated by dashes, are formed between protomers A and B.

Thus, the interaction between protomers occurs at multiple points across the contacting surfaces, resulting in the formation of dozens of weak bonds. This ensures that Contact surfaces bind with high specificity, virtually eliminating errors in the assembly of quaternary protein structures.

Complementarity is a universal principle of living systems that underlies the recognition and binding not only of protomers, but also of other (not necessarily protein) molecules.