ENERGETIC CONTRIBUTION OF SIDE-CHAIN HYDROGEN-BONDING TO THE STABILITY OF STAPHYLOCOCCAL NUCLEASE

Hydrogen bonds are a ubiquitous feature of protein structures, yet the re is great uncertainty about the energetic contribution of hydrogen b onding to protein stability. This study addresses this question by mak ing a series of single substitution mutations in the model protein sta phylococcal nuclease. These mutants have had a residue capable of part icipating in hydrogen bonding either removed or introduced. The varian ts we have investigated are as follows: nine valines substituted with threonine and serine; eight threonines converted to valine, serine, an d cysteine; and seven tyrosines replaced by phenylalanine and leucine. The stabilities of these 56 mutant proteins were determined by titrat ion with guanidine hydrochloride using fluorescence as a probe of stru cture. In general, it was found that the stability effects of removing a hydrogen bonding residue and replacing it with a nonbonding residue were relatively small. This was true even in the case of buried resid ues participating in hydrogen bonds, where the substituted residue lea ves an unfulfilled hydrogen bond in the hydrophobic core. In contrast, introducing a hydrogen bonding residue in place of a nonbonding resid ue was generally more costly energetically. A wide variability in the cost of burying a hydroxyl was observed, but this does not seem to be due to differences in hydrogen bonding. The overall energetic contribu tion of various wild-type hydrogen bonding interactions was evaluated as being favorable. A range of energies from approximately 1.5 to 4.0 kcal/mol was estimated for the contribution of these interactions to t he stability of the native state.