Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange

Douglas V Laurents; J Martin Scholtz; Manuel Rico; C Nick Pace; Marta Bruix

doi:10.1021/bi050142b

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Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange

Journal article

Peer reviewed

Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange

Douglas V Laurents, J Martin Scholtz, Manuel Rico, C Nick Pace and Marta Bruix

Biochemistry (Easton), Vol.44(21), pp.7644-7655

05/31/2005

DOI: 10.1021/bi050142b

PMID: 15909979

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Abstract

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 degrees C. In these conditions, the exchange mechanism was found to be EXII. The conformational stability calculated from the slowest exchanging amide groups was found to be 8.8 kcal/mol, in close agreement with values determined by spectroscopic methods. RNase Sa is curiously rich in acidic residues (pI = 3.5) with most basic residues being concentrated in the active-site cleft. The effects of dissolved salts on the stability of RNase Sa was studied by thermal denaturation experiments in NaCl and GdmCl and by comparing hydrogen exchange rates in 0.25 M NaCl to water. The protein was found to be stabilized by salt, with the magnitude of the stabilization being influenced by the solvent exposure and local charge environment at individual amide groups. Amide hydrogen exchange was also measured in 0.25, 0.50, 0.75, and 1.00 M GdmCl to characterize the 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 beta strands but in the 3/10 helix and an exterior beta strand. These structural elements are near the Cys7-Cys96 disulfide bond.

Amides - chemistry

Deuterium Exchange Measurement - methods

Disulfides - chemistry

Enzyme Stability

Guanidine - chemistry

Hot Temperature

Hydrogen-Ion Concentration

Isoenzymes - chemistry

Magnetic Resonance Spectroscopy

Protein Conformation

Protein Denaturation

Protein Folding

Protein Structure, Secondary

Protons

Ribonucleases - chemistry

Sodium Chloride - chemistry

Static Electricity

Streptomyces aureofaciens - enzymology

Thermodynamics

Details

Title: Subtitle: Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange
Creators: Douglas V Laurents - Texas A&M University
J Martin Scholtz - Texas A&M University
Manuel Rico - Texas A&M University
C Nick Pace - Texas A&M University
Marta Bruix - Texas A&M University
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.44(21), pp.7644-7655
DOI: 10.1021/bi050142b
PMID: 15909979
ISSN: 0006-2960
eISSN: 1520-4995
Grant note: GM 52483 / NIGMS NIH HHS GM 37039 / NIGMS NIH HHS
Language: English
Date published: 05/31/2005
Academic Unit: Research Administration; Pharmaceutical Sciences and Experimental Therapeutics; Biochemistry and Molecular Biology; Chemistry
Record Identifier: 9984288729702771

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