Hydrophobic core manipulations in ribonuclease T1

Differential scanning calorimetry, urea denaturation, and X-ray crystallogr aphy were combined to study the structural and energetic consequences of re filling an engineered cavity in the hydrophobic core of RNase T1 with CH3, SH, and OH groups. Three valines that cluster together in the major hydroph obic core of T1 were each replaced with Ala, Ser, Thr, and Cys. Compared to the wild-type protein, all these mutants reduce the thermodynamic stabilit y of the enzyme considerably. The relative order of stability at all three positions is as follows: Val > Ala approximate to Thr > Ser. The effect of introducing a sulfhydryl group is more variable. Surprisingly, a Val --> Cy s mutation in a hydrophobic environment can be as or even more destabilizin g than a Val --> Ser mutation. Furthermore, our results reveal that the pen alty for introducing an OH group into a hydrophobic cavity is roughly the s ame as the gain obtained from filling the cavity with a CH3 group. The inve rse equivalence of the behavior of hydroxyl and methyl groups seems to be c rucial for the unique three-dimensional structure of the proteins. The impo rtance of negative design elements in this context is highlighted.