Molecular Biology: Protein Structure and Functions - Stepanov V.M. 2005

Tertiary Protein Structure
The role of disulfide bonds in stabilizing the tertiary structure of certain proteins

Disulfide Bonds are formed through The oxidation of Cysteine residues that are brought into close proximity within the protein's Spatial Structure, converting them into cystine residues. As previously mentioned, these are the only type of covalent bonds that, alongside the non-covalent interactions discussed above, participate in stabilizing the tertiary structure. Disulfide bonds are by no means found in every protein; furthermore, highly stable—including thermostable—Proteins are known that completely lack them. Thus, the presence of disulfide bonds in a protein is by no means an absolute prerequisite for its stability, yet their role should not be underestimated.

THE CONTRIBUTION OF a disulfide bond to the conformational stability of a protein depends significantly on its Location within the spatial structure. Estimates obtained for A number of proteins (pancreatic Ribonuclease, Lysozyme, immunoglobulin light chains) place the contribution of disulfide bonds to the stabilization energy of the native spatial structure within the range of 2.3–5 kcal/mol. The Significance of these values becomes evident when compared to the relatively small stabilization energy of the protein globule, which is on the order of 10 kcal/mol. Apparently, the stabilization of Cell/13.html">Protein Structure by disulfide bonds is primarily explained by the fact that, by remaining intact in the denatured protein, they drastically reduce the number of possible configurations of the unfolded polypeptide chain, thereby lowering its Entropy.

As a consequence, the transition from such an unfolded chain containing disulfide bonds to the native compact structure entails a smaller decrease in conformational entropy and is consequently more favorable than in the absence of disulfide bonds. It is also probable that the preservation of covalent disulfide bonds in the unfolded chain between regions destined to be brought close together in the native globule facilitates the search for the correct folding pathway and has a favorable effect on The kinetics of protein molecule renaturation.

Obviously, both factors—those promoting the renaturation of the protein globule and those hindering its Denaturation—can only operate after the Spatial structure of the protein has formed. The formation of disulfide bonds during Biosynthesis does not precede the folding of the polypeptide chain into a globule, but rather accompanies this process, proceeding in parallel with it.

During the Formation of the protein globule, intermediate states likely arise where certain disulfide bonds are "incorrect," meaning they are formed between cysteine residues other than those linked in the native protein structure. Their "correction" may occur only at The final stage.

Experiments involving the Introduction of additional disulfide bonds into certain proteins via Protein Engineering Methods have shown that this does not always lead to an increase in protein stability.

In most cases, it is difficult to determine with absolute certainty why some proteins utilize disulfide bonds for stabilization, whereas others remain perfectly stable relying solely on non-covalent interactions. Apparently, disulfide bonds are important for the "kinetic" stability (resistance to external factors) of secretory proteins that function outside the relatively constant intracellular environment. Clearly, disulfide bonds—often multiple ones—are especially crucial for stabilizing small proteins that cannot form an extensive network of non-covalent interactions. Examples include many proteinase inhibitors, particularly the pancreatic Trypsin inhibitor. Its peptide chain, consisting of only 54 amino acid residues, is so well stabilized by four disulfide bonds that, despite its small size, this protein retains a compact pear-shaped form when heated up to 95°С.

However, these considerations cannot be regarded as rigid rules, as it is easy to point to secretory proteins, as well as small proteins, that lack disulfide bonds yet remain entirely stable.