Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange.
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.
Laurents
Douglas V
DV
Instituto de Química Física Rocasolano, CSIC, Serrano 119, Madrid 28006, Spain.
Scholtz
J Martin
JM
Rico
Manuel
M
Pace
C Nick
CN
Bruix
Marta
M
D000577:Amides / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011522:Protons; D012260:Ribonucleases / Q000737:chemistry*; D012965:Sodium Chloride / Q000737:chemistry; D055672:Static Electricity; D013304:Streptomyces aureofaciens / Q000201:enzymology; D013816:Thermodynamics
D000577:Amides; D004220:Disulfides; D007527:Isoenzymes; D011522:Protons; D012965:Sodium Chloride; D012260:Ribonucleases; C077002:ribonuclease Sa3; D019791:Guanidine