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
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1C54 2001-11-28 SOLUTION STRUCTURE OF RIBONUCLEASE SA
1T2H 2004-12-21 1.0 Y81W mutant of RNase Sa from Streptomyces aureofaciens
1LNI 2002-07-31 1.0 CRYSTAL STRUCTURE ANALYSIS OF A RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS AT ATOMIC RESOLUTION (1.0 A)
4GHO 2013-08-14 1.1 Crystal Structure Analysis of Streptomyces aureofaciens Ribonuclease S24A mutant
1T2I 2004-12-21 1.1 T76W mutant of RNase Sa from Streptomyces aureofaciens
1ZGX 2006-08-08 1.13 Crystal structure of ribonuclease mutant
1RGE 1996-10-14 1.15 HYDROLASE, GUANYLORIBONUCLEASE
1RGH 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
1RGF 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
1YNV 2005-07-19 1.2 Asp79 makes a large, unfavorable contribution to the stability of RNase Sa
1RGG 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
4J5K 2014-05-28 1.23 Crystal structure analysis of Streptomyces aureofaciens ribonuclease Sa Y51F mutant
1I8V 2001-09-19 1.25 CRYSTAL STRUCTURE OF RNASE SA Y80F MUTANT
4J5G 2014-05-28 1.31 Crystal structure analysis of Streptomyces aureofaciens ribonuclease Sa T95A mutant
1BOX 1999-12-29 1.6 N39S MUTANT OF RNASE SA FROM STREPTOMYCES AUREOFACIENS
3A5E 2010-08-04 1.6 Crystal structure of 5K RNase Sa
1GMP 1993-10-31 1.7 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1AY7 1999-03-02 1.7 RIBONUCLEASE SA COMPLEX WITH BARSTAR
1I70 2001-09-19 1.7 CRYSTAL STRUCTURE OF RNASE SA Y86F MUTANT
1GMR 1993-10-31 1.77 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1UCK 2003-09-09 1.8 Mutants of RNase Sa
1SAR 1992-04-15 1.8 DETERMINATION AND RESTRAINED LEAST-SQUARES REFINEMENT OF THE CRYSTAL STRUCTURES OF RIBONUCLEASE SA AND ITS COMPLEX WITH 3'-GUANYLIC ACID AT 1.8 ANGSTROMS RESOLUTION
1GMQ 1993-10-31 1.8 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
2SAR 1992-04-15 1.8 DETERMINATION AND RESTRAINED LEAST-SQUARES REFINEMENT OF THE CRYSTAL STRUCTURES OF RIBONUCLEASE SA AND ITS COMPLEX WITH 3'-GUANYLIC ACID AT 1.8 ANGSTROMS RESOLUTION
1UCI 2003-09-09 1.8 Mutants of RNase Sa
1UCJ 2003-09-09 1.81 Mutants of RNase Sa
1UCL 2003-09-09 1.82 Mutants of RNase Sa
1RSN 1995-12-07 2.0 RIBONUCLEASE (RNASE SA) (E.C.3.1.4.8) COMPLEXED WITH EXO-2',3'-CYCLOPHOSPHOROTHIOATE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Guanyl-specific ribonuclease Sa P05798 RNSA_KITAU