Ribonuclease Sa Conformational Stability Studied by NMR-Monitored Hydrogen Exchange

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• 作者：Douglas V. Laurents ; J. Martin Scholtz ; Manuel Rico ; C. Nick Pace ; and Marta Bruix
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：May 31, 2005
• 年：2005
• 卷：44
• 期：21
• 页码：7644 - 7655
• 全文大小：395K
• 年卷期：v.44,no.21(May 31, 2005)
• ISSN：1520-4995

文摘

The conformational stability of ribonuclease Sa (RNase Sa) has been measured at the per-residue level by NMR-monitored hydrogen exchange at pH* 5.5 and 30 C. In these conditions, theexchange mechanism was found to be EXII. The conformational stability calculated from the slowestexchanging amide groups was found to be 8.8 kcal/mol, in close agreement with values determined byspectroscopic methods. RNase Sa is curiously rich in acidic residues (pI = 3.5) with most basic residuesbeing concentrated in the active-site cleft. The effects of dissolved salts on the stability of RNase Sa wasstudied by thermal denaturation experiments in NaCl and GdmCl and by comparing hydrogen exchangerates in 0.25 M NaCl to water. The protein was found to be stabilized by salt, with the magnitude of thestabilization being influenced by the solvent exposure and local charge environment at individual amidegroups. Amide hydrogen exchange was also measured in 0.25, 0.50, 0.75, and 1.00 M GdmCl to characterizethe unfolding events that permit exchange. In contrast to other microbial ribonucleases studied to date,the most protected, globally exchanging amides in RNase Sa lie not chiefly in the central strands butin the 3/10 helix and an exterior strand. These structural elements are near the Cys7-Cys96 disulfidebond.