Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange.


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. Study holds ProTherm entries: 19308, 19309, 19310, 19311, 19312, 19313, 19314, 19315, 19316, 19317, 19318, 19319, 19320, 19321, 19322, 19323, 19324, 19325, 19326, 19327, 19328, 19329, 19330, 19331, 19332, 19333, 19334, 19335, 19336, 19337, 19338, 19339, 19340, 19341, 19342, 19343, 19344, 19345, 19346, 19347, 19348, 19349, 19350, 19351, 19352, 19353, 19354, 19355, 19356, 19357, 19358, 19359, 19360, 19361, 19362, 19363, 19364, 19365, 19366, 19367, 19368, 19369, 19370, 19371, 19372, 19373, 19374, 19375, 19376, 19377, 19378, 19379, 19380, 19381, 19382, 19383, 19384, 19385, 19386, 19387, 19388, 19389, 19390, 19391, 19392, 19393, 19394, 19395, 19396, 19397, 19398 Extra Details: Experiments were carried out on the amide group of Val2. The data is correspond to Figure 6 of the article and received through personal correspondence. conformational stability; hydrogen exchange; amide groups; salts; beta strands; disulfide bond

Submission Details

ID: AgEsyqXq3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Laurents DV;Scholtz JM;Rico M;Pace CN;Bruix M,Biochemistry (2005) Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange. PMID:15909979
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