16] Stabilization of functional proteins by introduction of multiple disulfide bonds

Abstract

Publisher Summary This chapter discusses the stabilization of functional proteins by the introduction of multiple disulfide bonds. There are at least three aspects to be considered in the overall design and introduction of disulfide bonds for the stabilization of proteins: the geometric requirements for the formation of disulfide bonds, the size of the loop formed by the cross-link, and the loss of existing interactions associated with replacement of wild-type residues by cysteines. The effect of disulfide bonds on the stability of proteins are presumed to be due to a decrease in the conformational chain entropy of the unfolded polypeptide. This large stabilizing potential has made the engineering of disulfide bonds into proteins a strategy for the improvement of protein stability. Disulfide bonds have been introduced in dihydrofolate reductase, phage T4 lysozyme, subtilisin, and λ repressor. In all cases, the newly introduced cysteines are found to form disulfide bonds. However, the addition of these new disulfides did not always confer an increase in stability. One of the reasons for this modest success appears to be the difficulty in finding optimal sites that are compatible with the strict stereochemical requirements of disulfide bridges. The introduction of an atypical disulfide can introduce strain into the protein structure and can offset the stabilizing effect of the cross-link. Recently a number of single and multiple disulfide bonds were introduced in phage T4 lysozyme and have shown that some of the variants are significantly more stable than wild type. This chapter also describes the design, construction, and characteristics of these mutant proteins.