ENGINEERED DISULFIDE BONDS IN STAPHYLOCOCCAL NUCLEASE - EFFECTS ON THE STABILITY AND CONFORMATION OF THE FOLDED PROTEIN

Efforts to enhance the stability of proteins by introducing engineered disulfide bonds have resulted in mixed success. Most approaches to th e prediction of the energetic consequences of disulfide bond formation in proteins have considered only the destabilizing effects of cross-l inks on the unfolded state (chain entropy model) [Pace, C. N., Grimsle y, G. R., Thomson, J. A., & Barnett, B. J. (19S8) J. Biol. Chem. 263, 11820-11825; Doig, A. J., & Williams, D. H. (1991) J. Mol. Biol. 217, 389-398]. It seems clear, however, that disulfide bridges also can inf luence tile stability of the native state. In order to assess the impo rtance of the latter effect, we have studied four variants of staphylo coccal nuclease (VS strain) each containing one potential disulfide br idge created by changing two wild-type residues to cysteines by site-d irected mutagenesis. In each case, one of the introduced cysteines was within the type VIa beta turn containing cis Pro(117), and the other was located in the adjacent extended loop containing Gly(79) In all fo ur cases, the overall loop size was kept nearly constant (the number o f residues in the loop between the two cysteines varied from 37 to 42) so as to minimize differences from chain entropy effects. The objecti ve was to create variants in which a change in the reduction state of tile disulfide would be coupled to a change in the position of the equ ilibrium between file cis and trans forms of the Xxx(116)-Pro(117) pep tide bond in the folded slate of the protein. The position of this equ ilibrium, which can be detected by NMR spectroscopy, has been shown pr eviously to correlate with the stability of the native protein. Its de termination provides a measure of strain in the folded state. The ther mal stabilities and free energies for unfolding by elevated temperatur e and guanidinium chloride were measured for each of the four mutants under conditions in which the Introduced cysteines were cross-linked ( oxidized) and unlinked (reduced). In addition: reduction potentials we re determined for each mutant. Formation of the different disulfide br idges was found to induce varying levels of folded state strain. The s tabilization energy of a given disulfide bridge could be predicted fro m the measured perturbation energy for the peptide bond isomerization, provided that energetic effects on the unfolded state were calculated according to the chain entropy model. Undiagnosed strain in native sl ates of proteins may explain the variability observed in the stabiliza tion provided by engineered disulfide bridges.