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

CHAPTER 1. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

III. Formation of the Three-Dimensional Protein Structure in the Cell

The formation of the three-dimensional Structure of Proteins is a critical biological process, as their spatial architecture directly determines their biological function.

The process by which a polypeptide chain folds into its correct Spatial Structure is known as protein folding. Individual proteins, being products of the same Gene, share an identical Amino Acid Sequence and acquire the exact same conformation and function under identical cellular conditions. This principle is supported by the ability of certain proteins, following Denaturation (which disrupts weak bonds without damaging the Primary Structure), to spontaneously restore their unique conformation and function.

However, the concentration of proteins within The Cell is so high that There is a significant probability of interaction among proteins with unformed Conformations. Their surfaces expose hydrophobic radicals that tend to aggregate. Consequently, for many high-molecular-weight proteins with complex spatial structures, folding occurs with the participation of a special group of proteins called chaperones (from the French chaperon — duenna or nurse).

A. Protein renaturation

For a long time, Protein Denaturation was considered irreversible. However, it has been demonstrated that certain purified and denatured proteins can regain their native conformation under experimental conditions once the Denaturing Agents are removed.

Renaturation of Ribonuclease

In the early 1960s, it was discovered that protein denaturation can be reversible. This breakthrough came from studying the denaturation of ribonuclease—an enzyme that cleaves bonds between NUCLEOTIDES in RNA. Ribonuclease is a globular protein consisting of a single polypeptide chain of 124 amino acid residues. Its conformation is stabilized by 4 Disulfide Bonds and numerous weak interactions.

Treatment of ribonuclease with β-mercaptoethanol (formula: НО-СН₂-СН₂- SН) leads to the reduction of disulfide bonds and the restoration of Cysteine SH-groups, thereby disrupting the compact Protein Structure. The addition of an 8 M urea solution or a 6 M guanidine hydrochloride solution—agents that break weak bonds within the protein and form new Hydrogen Bonds with the denaturants—results in randomly coiled polypeptide chains devoid of enzymatic activity, i.e., denaturation of the enzyme. Denaturing agents do not disrupt the Introduction/19.html">Primary structure of the protein.

Nevertheless, if ribonuclease is freed from denaturing agents and β-mercaptoethanol via dialysis, its enzymatic activity is gradually restored. This process is referred to as protein renaturation or renatuation. The sulfhydryl groups of the denatured enzyme are oxidized by atmospheric oxygen, spontaneously re-forming the 4 disulfide bonds characteristic of the native protein structure. Out of 105 possible ways to link the eight cysteine SH-groups, only the single variant characteristic of the native conformation is realized (Fig. 1-22).

Class="center">Fig. 1-22. Denaturation and renaturation of ribonuclease. A — native ribonuclease molecule featuring 4 disulfide bonds in its tertiary structure; Б — denatured ribonuclease molecule; В — native ribonuclease molecule with the 4 disulfide bonds successfully re-established between the same cysteine residues.

The possibility of renaturation was subsequently proven for other proteins, notably Myoglobin. The preservation of the protein's primary structure is a prerequisite for the recovery of its conformation. These experiments led to the formulation of a fundamental principle of molecular biology: The amino acid sequence of a protein dictates its conformation and specific function.

The formation of a protein's spatial structure is a spontaneous process wherein the protein tends to adopt the conformation of lowest Free energy under given conditions. Alterations in the environmental conditions or Changes in the primary structure of a given protein can lead to shifts in its conformation and function.

B. Structure and Functional Role of Chaperones in Protein Folding

During the synthesis of polypeptide chains, their membrane transport, and the assembly of Oligomeric Proteins, unstable intermediate conformations prone to aggregation inevitably arise. Newly synthesized Polypeptides expose numerous hydrophobic radicals that are normally buried within the core of the three-dimensional structure. Therefore, while the native conformation is being established, the reactive amino acid residues of certain proteins must be shielded from interacting with identical groups on other proteins.

Proteins capable of binding to unstable, aggregation-prone polypeptide states have been discovered in all known organisms, From Prokaryotes to higher eukaryotes. By stabilizing their conformation, these proteins facilitate proper folding and have been designated as chaperones.

1. Classification of Chaperones

Based on their molecular weight, all chaperones can be divided into 6 main groups:

✵ High-molecular-weight chaperones, with molecular weights ranging from 100 to 110 kDa;

✵ Hsp90 — with molecular weights ranging from 83 to 90 kDa;

✵ Hsp70 — with molecular weights ranging from 66 to 78 kDa;

✵ Hsp60;

✵ Ch-40;

✵ low-molecular-weight chaperones with a molecular weight ranging from 15 to 30 kDa.

Chaperones are classified into: constitutive proteins (whose high basal synthesis is independent of cellular stress) and inducible proteins, whose synthesis is low under normal conditions but increases sharply in response to cellular stress. Inducible proteins are classified as "heat Shock proteins," which are rapidly synthesized in virtually all Cells exposed to any form of stress. The name "heat shock proteins" originated because these proteins were first discovered in cells subjected to high temperatures.

2. The Role of chaperones in protein folding

During Protein Synthesis, the N-terminal region of the polypeptide is synthesized before the C-terminal region. The complete amino acid sequence is required for the Formation of the protein's conformation. Therefore, while the protein is being synthesized on the ribosome, Ch-70 protect reactive radicals (especially hydrophobic ones).

Ch-70 is a highly conserved class of proteins present in all cellular compartments: the Cytoplasm, Nucleus, ER, and Mitochondria. Near the carboxy-terminus of the single polypeptide chain of chaperones, there is a groove-like region formed by amino acid radicals. It can interact with segments of protein molecules and unfolded polypeptide chains 7 to 9 Amino Acids long, enriched in hydrophobic radicals. In a nascent polypeptide chain, such segments appear approximately every 16 amino acids.

The folding of many high-molecular-weight proteins with complex conformations (such as those with a domain structure) takes place within a specialized space formed by Ch-60. Ch-60 Functions as an oligomeric complex consisting of 14 subunits (Fig. 1-23).

Fig. 1-23. STRUCTURE OF THE chaperone complex consisting of 14 Ch-60 protein molecules.

Ch-60 forms 2 rings, each consisting of 7 interconnected subunits. A Ch-60 subunit comprises 3 domains: apical, intermediate, and equatorial. The apical domain features a series of hydrophobic residues facing the inner cavity of the ring formed by the subunits. The equatorial domain contains an ATP-binding site and exhibits ATPase activity, meaning it is capable of hydrolyzing ATP to ADP and H₃PO₄.

The chaperone complex exhibits high affinity for proteins whose surfaces display features typical of unfolded molecules (primarily regions enriched in hydrophobic radicals). Upon entering the cavity of the chaperone complex, the protein binds to the hydrophobic radicals of the apical regions of Ch-60. Within the specific environment of this cavity, isolated from other cellular molecules, various potential protein conformations are sampled until the single energetically most favorable conformation is found.

The release of the protein with its formed native conformation is accompanied by ATP Hydrolysis in the equatorial domain. If the protein fails to acquire its native conformation, it re-binds to the chaperone complex. Such chaperone-dependent protein folding requires a substantial expenditure of energy.

Thus, PROTEIN SYNTHESIS AND folding occur with the participation of different groups of chaperones, which prevent unwanted interactions between proteins and other cellular molecules and chaperone them until their native structure is fully formed (Fig. 1-24).

Fig. 1-24. Participation of chaperones in protein folding. A — involvement of HSP70 chaperones in preventing hydrophobic interactions between segments of the nascent polypeptide; B — formation of the native protein conformation within the chaperone complex.

3. The role of chaperones in protecting cellular proteins from denaturing stress

As mentioned above, chaperones involved in protecting cellular proteins from denaturing conditions are classified as heat shock proteins (HSPs).

When cells are exposed to various stress factors (high Temperature, Hypoxia, infection, UV radiation, changes in medium pH or molarity, toxic chemicals, heavy metals, etc.), the synthesis of HSPs increases. Possessing a high affinity for the hydrophobic regions of partially denatured proteins, they can prevent complete denaturation and restore the native conformation of the proteins.

Research has shown that brief exposure to stress increases HSP production and enhances the body's resistance to prolonged stress. For instance, short-term myocardial ischemia during moderate exercise significantly increases myocardial resistance to prolonged ischemia caused by angina or coronary thrombosis. Currently, searching for pharmacological and molecular-biological ways to activate HSP synthesis in cells is considered a promising avenue in medical research.

4. DISEASES ASSOCIATED WITH protein misfolding

Calculations have shown that only a small fraction of theoretically possible polypeptide chain variants can adopt a single stable spatial structure. Most such proteins can assume multiple conformations with approximately equal Gibbs free energy but distinct properties. The primary structure of most known evolutionarily selected proteins ensures the exceptional stability of a single conformation.

However, under altered conditions, certain Water-soluble proteins can acquire the conformation of poorly soluble, aggregation-prone molecules that form fibrillar deposits in cells known as amyloid (from Lat. amylum — starch). Just like starch, amyloid deposits can be detected by staining Tissues with iodine. This can occur:

✵ due to the overproduction of certain proteins, resulting in an increased concentration within the cell;

✵ upon the introduction into cells or the formation within them of proteins capable of influencing the conformation of other protein molecules;

✵ upon the activation of normal cellular protein proteolysis, leading to the formation of insoluble fragments prone to aggregation;

✵ As a result of point Mutations in the protein structure.

The deposition of amyloid in Organs and tissues disrupts cellular Structure and function, leading to degenerative changes and the proliferation of Connective Tissue or glial cells. This gives rise to disorders known as amyloidoses. Each type of amyloidosis is characterized by a specific type of amyloid. To date, more than 15 such diseases have been described.

Alzheimer's disease

Alzheimer's disease is the most common β-amyloidosis of The Nervous system, typically affecting elderly individuals and characterized by progressive memory impairment and severe personality degradation. The condition involves the deposition of β-amyloid in Brain tissue—a protein that forms insoluble fibrils that disrupt the structure and function of Nerve Cells. β-Amyloid results from a conformational alteration of a normal human protein. It is generated from a larger precursor via partial proteolysis and is synthesized in various tissues. Unlike its normal precursor, which is rich in α-helical regions, β-amyloid adopts a secondary β-pleated sheet structure, aggregates into insoluble fibrils, and is resistant to Proteolytic Enzymes.

The exact causes of native protein folding impairment in brain tissue remain to be elucidated. It is hypothesized that Aging may lead to a decline in the synthesis of chaperones—proteins responsible for assisting in the formation and maintenance of native protein conformation—or an increase in protease activity, resulting in elevated concentrations of proteins prone to conformational changes.

Prion Diseases

Prions are a unique class of proteins endowed with infectious properties. Upon entering The Human Body or arising spontaneously within it, they can trigger severe, incurable Central Nervous System disorders known as prion diseases. The term "prion" is derived from the acronym for the English phrase "proteinaceous infectious particle."

The prion protein is encoded by the same gene as its normal counterpart, meaning they share an identical primary structure. However, the two proteins exhibit distinct conformations: the prion protein is characterized by a high content of β-sheets, whereas the normal protein contains numerous α-helical domains. Furthermore, the prion protein is resistant to protease action and, upon entering brain tissue or forming there spontaneously, promotes The conversion of the normal protein into the prion form through Protein-Protein Interactions. This creates a so-called "polymerization nucleus" composed of aggregated prion proteins, to which new molecules of the normal protein can attach. As a result, conformational rearrangements characteristic of prion proteins occur in their spatial structure.

Cases of hereditary forms of prion diseases caused by mutations in The structure of this protein are well documented. However, human infection with prion proteins is also possible, leading to a fatal disease. For instance, kuru is a prion disease indigenous to the aborigines of New Guinea, where its epidemic spread was linked to traditional ritual cannibalism within these tribes and the transmission of the infectious protein from one individual to another. Due to lifestyle changes, this disease has now practically vanished.

Currently, interest in prion diseases has surged due to human infections resulting from the consumption of meat products derived from animals carrying prions that cause bovine spongiform encephalopathy, or "mad cow disease" (Creutzfeldt-Jakob disease). Although Human and Animal prion proteins differ only slightly, it was long believed that interspecies barriers hindered disease transmission. Recent findings, however, have demonstrated that these barriers are not absolute and that cross-species transmission is entirely plausible. For example, by mid-1999, approximately 40 cases of this disease had been registered in the United Kingdom. Epidemiological projections do not rule out the potential development of a prion disease epidemic over the next 10 to 15 years.