Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix.

TitleHydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix.
Publication TypeJournal Article
Year of Publication1999
AuthorsHuyghues-Despointes, B. M., U. Langhorst, J. Steyaert, C. N. Pace, and J. M. Scholtz
JournalBiochemistry
Volume38
Issue50
Pagination16481-90
Date Published1999 Dec 14
ISSN0006-2960
KeywordsAlanine, Amino Acid Substitution, Crystallography, X-Ray, Glycine, Hydrogen, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Denaturation, Protein Structure, Secondary, Ribonuclease T1, Solvents, Urea
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 (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O 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 DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), 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.

Alternate JournalBiochemistry
PubMed ID10600109
Grant ListGM37039 / GM / NIGMS NIH HHS / United States
GM52483 / GM / NIGMS NIH HHS / United States