Hydrogen-Exchange Stabilities of RNase T1 and Variants with Buried and Solvent-Exposed Ala → Gly Mutations in the Helix

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Abstract
Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala → Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (ΔGHX) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (ΔGU), if the effects of D2O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol.6, 210−212]. These residues also show similar changes in ΔGHX upon Ala → Gly mutations (ΔΔGHX) as compared to equilibrium measurements (ΔΔGU), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci.U.S.A.94, 3833−2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala → Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.