Lysozyme engineered disulfide bonds

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. 

Perry, L.J. Wetzel, R. Disulfide bond engineered into T4 lysozyme stabilization of the protein toward thermal inactivation. Science 1984, 226 (4674), 555-557. 

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. 

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