BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 5. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

5.2. Protein Structure

5.2.3. Tertiary Protein Structure

The Tertiary Structure of a protein is its three-dimensional spatial conformation formed through interactions between amino acid side chains (radicals) that may be located at considerable distances from each other along the polypeptide chain.

Bonds involved in The formation of Tertiary Cell/13.html">Protein Structure. During folding, a protein's polypeptide chain tends to adopt an energetically favorable conformation characterized by a minimum of Free energy. Therefore, hydrophobic amino acid residues tend to cluster within the interior of Water-soluble Globular Proteins. This leads to the formation of so-called hydrophobic interactions, as well as Van der Waals forces between closely spaced atoms. As a result, a Hydrophobic core forms inside the protein globule. During Secondary structure formation, the hydrophilic groups of the peptide backbone form A large number of Hydrogen Bonds, thereby preventing water binding and the disruption of the tight internal protein structure.

Hydrophilic amino acid residues tend to form hydrogen bonds with water and are therefore primarily located On the surface of the protein molecule. All hydrophilic groups of amino acid side chains that end up inside the hydrophobic core interact with one another via ionic and hydrogen bonds (Fig. 5.11).

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Fig. 5.11. Types of bonds that occur between amino acid side chains during the formation of tertiary protein structure:

1 - ionic; 2 - hydrogen; 3 - hydrophobic; 4 — disulfide

Ionic bonds can form between negatively charged (anionic) carboxyl groups of aspartic and

glutamic acid side chains and positively charged (cationic) groups of Arginine, Lysine, or Histidine side chains.

Hydrogen bonds form between hydrophilic uncharged groups (such as -OH, -CONH₂, SH groups) and any other hydrophilic groups.

Proteins functioning in a non-polar (lipid) environment, such as Membrane Proteins, have a different architecture: hydrophilic amino acid residues are located in the interior of the protein, whereas hydrophobic Amino Acids are localized on the molecular surface and contact the non-polar environment. In each case, The amino acid side chains assume the most bioenergetically favorable position.

The tertiary structure of certain proteins is stabilized by Disulfide Bonds formed through the interaction of SH groups from two Cysteine residues. These two cysteine residues may be located far apart in the linear Introduction/19.html">Primary Structure of the protein, but during tertiary structure formation, they come into close proximity and form a strong covalent bond between their side chains (Fig. 5.12).

Fig. 5.12. Formation of a disulfide bond in proteins

Most intracellular proteins lack disulfide bonds. However, such bonds are common in proteins secreted by Cells into the extracellular space. It is believed that these covalent bonds stabilize protein conformation outside The Cell and prevent Denaturation. Examples include the hormone Insulin and IMMUNOGLOBULINS.

Insulin is a protein hormone containing 51 amino acids, consisting of two polypeptide chains (chain A contains 21 amino acids, chain B contains 30 amino acids). Insulin is synthesized in the pancreatic β-cells and secreted into the bloodstream in response to elevated Blood glucose levels. The structure of insulin features two disulfide bonds connecting the two polypeptide chains A and B, and one intra-chain disulfide bond within chain A (Fig. 5.13).

All proteins with the identical primary structure placed under identical 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 its native structure.

Fig. 5.13. Disulfide bonds in the STRUCTURE OF THE insulin hormone

Hydrophobic interactions, as well as ionic and hydrogen bonds, are classified as weak bonds because their energy is only slightly higher than the energy of thermal atomic motion at room Temperature (disruption of such bonds is possible even at this temperature). Maintaining a protein's characteristic conformation is made possible by the formation of a vast number of weak bonds between different Regions of the polypeptide chain.

Proteins consist of a tremendous number of atoms in constant (Brownian) motion, which leads to minor displacements of individual regions of the polypeptide chain that generally do not disrupt the overall Protein Structure and its function. Consequently, proteins exhibit conformational lability—a tendency to undergo minor conformational changes through the breaking of some weak bonds and the formation of others. Protein conformation can change in response to alterations in the chemical and Physical Properties of the environment, as well as upon protein interaction with other molecules. As a result, changes occur in the Spatial Structure not only of the region contacting the other molecule, but also of the protein conformation as a whole. Conformational changes play a crucial role in protein function within a living cell.

The rupture of a large number of weak bonds within a protein molecule leads to The breakdown of its conformation. Because bond breakage caused by various factors is random in nature, molecules of a single individual protein in solution assume the form of randomly coiled, disordered structures that differ from one another in their three-dimensional arrangement. The loss of native conformation is accompanied by the loss of specific protein function. This process is called Protein Denaturation. No Cleavage of peptide bonds occurs during protein denaturation, meaning the Primary Protein Structure remains intact.

In a denatured protein, hydrophobic residues that are normally hidden within the hydrophobic core in the Native State become exposed on the surface. At a sufficiently high protein concentration and in the absence of a strong repulsive charge, these molecules can aggregate via hydrophobic interactions, leading to decreased solubility and subsequent precipitation.

During denaturation, the compact and dense three-dimensional structure of a native protein rapidly expands, becoming readily accessible for peptide bond cleavage by Proteolytic Enzymes (Fig. 5.14). Cooking meat not only improves its flavor but also facilitates its Enzymatic Digestion in the gastrointestinal tract. Furthermore, the acidic environment of The Stomach exerts a denaturing effect on dietary proteins that have not undergone prior thermal Processing, as well as on the proteins of microorganisms ingested with food.

Protein denaturation is caused by factors that disrupt the hydrophobic, hydrogen, and ionic Bonds Responsible for stabilizing the protein conformation:

✵ high temperatures (> 50 °C), which increase the thermal motion of atoms and molecules, leading to the cleavage of weak bonds;

✵ vigorous agitation of the solution, which causes protein molecules to collide with the air at the phase boundary, altering their conformation;

✵ organic substances (e.g., ethanol, phenol, and its derivatives) capable of interacting with Functional groups of proteins, thereby inducing conformational changes. In biochemical research, urea or guanidine hydrochloride are frequently used to denature proteins; these agents form hydrogen bonds with the amino and carbonyl groups of the peptide backbone and Certain amino acid side-chain functional groups. This disrupts the bonds involved in maintaining the secondary and tertiary structures of native proteins and leads to the formation of new bonds with the chemical Reagents:

Fig. 5.14. Structure of a native protein molecule (center) and three denatured molecules of the same protein

✵ heavy metal salts (copper, mercury, silver, lead, etc.), which form strong bonds with essential protein functional groups (most commonly -SH groups), altering their conformation and activity;

✵ detergents, which are substances containing a hydrophobic hydrocarbon radical and a hydrophilic functional group (known as amphiphilic substances). The hydrophobic residues of proteins interact with the hydrophobic parts of detergents, altering the protein conformation. Denatured by the action of detergen-

ts, the protein typically remains in solution because the hydrophilic portions of the denaturing agent keep it solubilized. Common examples of detergents include various soaps (Fig. 5.15).

Fig. 5.15. Protein denaturation by detergents

The susceptibility of most proteins to denaturation during isolation, storage, and handling significantly complicates their production and application in medicine.

To ensure proper handling of protein-based Pharmaceuticals, manufacturers provide instructions detailing storage and usage conditions. For instance, most protein drugs must be stored at temperatures not exceeding 10 °C, and lyophilized (dry) preparations should be reconstituted with boiled water cooled to room temperature to prevent denaturation.

In biochemical studies, proteins are typically removed from solution prior to the determination of low-molecular-weight compounds in biological samples. Trichloroacetic acid is best suited for this purpose. Upon its addition to the solution, the denatured proteins precipitate and are easily removed by filtration. Trichloroacetic acid can also be used to denature enzymes in order to halt an enzymatic reaction.

In medical practice, Denaturing Agents are frequently employed for the sterilization of surgical instruments and Materials, as well as antiseptics. For example, medical instruments and materials are sterilized in an autoclave using high temperature.

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 cellular proteins. The antimicrobial efficacy decreases as the solubility of the compound in water increases.

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

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

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

Strong antimicrobial activity is exhibited, for example, by corrosive sublimate—mercury(II) chloride (HgCl₂). It is used for hand antisepsis and room disinfection. Accidental or intentional poisoning with mercury compounds causes severe necrotic lesions of the gastrointestinal mucosa and necrotic Changes in the Kidneys. Silver preparations, such as silver nitrate (AgNO₃) and collargol (colloidal silver), also possess antimicrobial properties and are used to treat mucous membranes during infectious diseases.