Disulfide protein stability

Disulfides. The introduction of disulfide bonds can have various effects on protein stability. In T4 lyso2yme, for example, the incorporation of some disulfides increases thermal stability others reduce stability (47—49). Stabili2ation is thought to result from reduction of the conformational entropy of the unfolded state, whereas in most cases the cause of destabili2ation is the introduction of dihedral angle stress. In natural proteins, placement of a disulfide bond at most positions within the polypeptide chain would result in unacceptable constraint of the a-carbon chain. 

Matsumura, M., Signor, G., Matthews, B.W. Substantial increase of protein stability by multiple disulfide bonds. Nature 342 291-293, 1989. 

Some proteins contain covalent disulfide (S— S) bonds that link the sulfhydryl groups of cysteinyl residues. Formation of disulfide bonds involves oxidation of the cysteinyl sulfhydryl groups and requires oxygen. Intrapolypeptide disulfide bonds further enhance the stability of the folded conformation of a peptide, while interpolypeptide disulfide bonds stabilize the quaternary structure of certain oligomeric proteins. 

The extrusion process frequently results in realignment of disulfide bonds and breakage of intramolecular bonds. Disulfide bonds stabilize the tertiary structure of protein and may limit protein imfolding during extrusion (Taylor et al., 2006). Flow and melt characteristics were improved when other proteins were extruded with disulfide reducing agents (Areas, 1992), which indicates that disulfide bonds adversely affect... 

The native conformation of proteins is stabilized by a number of different interactions. Among these, only the disulfide bonds (B) represent covalent bonds. Hydrogen bonds, which can form inside secondary structures, as well as between more distant residues, are involved in all proteins (see p. 6). Many proteins are also stabilized by complex formation with metal ions (see pp. 76, 342, and 378, for example). The hydrophobic effect is particularly important for protein stability. In globular proteins, most hydrophobic amino acid residues are arranged in the interior of the structure in the native conformation, while the polar amino acids are mainly found on the surface (see pp. 28, 76). 

PROTEIN DISULFIDE ISOMERASE STABILITY CONSTANT METAL ION COMPLEXATION... 

Thus we note that cations (and anions) in proteins would have a very general effect on protein stability, either through cross-linking (similar to the effects of disulfide bridges) or through a general electrostatic effect... 

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

At present, 16 cysteine-containing subtilisin-type enzymes are known and the position of the cysteine residues is restricted to the nine corresponding sites described above.42 Of the 16 enzymes, six enzymes other than aqualysin I and proteinase K have cysteine residues at positions where the cysteine residues are able to form disulfide bond(s) like the two enzymes. Although these disulfide bonds seem to have been acquired to increase protein stability, only four kinds of disulfide bonds are found in the subtilisin-type enzymes, suggesting that the positions of the disulfide bonds have been selected strictly in the process of molecular evolution of the enzyme. 

Because of the strict stereochemical requirements, it is not easy to find optimal sites for the introduction of disulfide bonds into proteins. Introduction of disulfide bonds into T4 lysozyme has been engineered by theoretical calculations and computer modeling.4 7 The results obtained from the mutant lysozymes illustrate several points relevant to the use of disulfide bonds for improving protein stability.6 (i) Introduction of the cysteine(s) should minimize the disruption or loss of interactions that stabilize the native structure, (ii) The size of the loop formed by the crosslink should be as large as possible, (iii) The strain energy introduced by the disulfide bond should be kept as low as possible. For this purpose, a location within the flexible part of the molecule is desirable. 

If certain amino acids (such as disulfide bonds or crucial amino acids in the hydrophobic core) are indispensable for protein stability, these positions can be changed by site-directed mutagenesis (Proba et al., 1998). To avoid back-mutations during the evolution process or the selection of a residual wild-type contamination, the pool is amplified after each round of ribosome display with a primer that reintroduces the destabilizing mutation. If the mutation is not close to one of the termini, the coding sequence has to be amplified in two parts, which are then reassembled by PCR. Thus, to evolve improved stabilities this strategy first removes known crucial stabilizing factors to select for compensatory mutations at different positions. 

Peptides that form a helices that associate as coiled coils, or as three- or four-helix tetrameric bundles or amphipathic helices that associate with lipid bilayers have been made. More difficult has been the design of proteins that form (3 sheets. Many efforts are being made to xmderstand protein stability " by systematic substitutions of one residue for another. Addition of new disulfide linkages at positions selected by study of three-dimensional structures sometimes stabilizes enzymes. On the other hand elimination of unnecessary cysteine residues can enhance stability by preventing p elimination and replacement of asparagine by threonine can improve the thermostability of enzymes by preventing deami-dation. ... 

Rational methods are based on experimental evidence that the effect of single amino acid substitutions on protein stability can be well approximated as additive, distributed, and large independent interactions. Moreover, by comparison of homologous enzymes from thermophilic and nonthermophilic microorganisms, it is known that introduction of disulfide bridges as well as increased numbers of proline residues increase protein stability. 

The combination of molecular modeling with genetic engineering to enhance protein stability has been successful in certain cases. For instance, introducing carefully sited novel disulfide bonds increased protein stability in T4 lysozyme (11-13) and in X-repressor (14). However, the results in other proteins, for instance, in subtilisin (15,16) and in dihydrofolate reductase (17) have been less predictable. 

The key variables affecting protein stability in the refolding process are temperature, pH, ionic strength, denaturant concentration and protein concentration (5). In proteins stabilized by disulfide bonds in the native state, additional variables include dissolved oxygen, trace metal ion and reducing agent concentrations (6). 

Considering chemical stability, even alterations at single amino acids or the peptide bond can be detrimental [6], Chemical reactions having an impact on protein stability include hydrolysis of the peptide bond, deamidation, oxidation, p-elimina-tion, isomerization, and disulfide bond breakage and formation. The extent to which they occur is mainly influenced by the temperature and pH value of the solution [24], Bearing in mind that proteins react sensitively to the above-mentioned environmental conditions, preparation procedures for protein pharmaceuticals have to be chosen very carefully to preserve protein integrity and functionality. 

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