Increasing protein stability: importance of DeltaC(p) and the denatured state.


Abstract

Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the beta-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28 degrees C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, DeltaC(p), by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles. Study holds ProTherm entries: 25631, 25632, 25633, 25634, 25635, 25636, 25637, 25638, 25639, 25640, 25641, 25642, 25643, 25644, 25645, 25646, 25647, 25648, 25649, 25650, 25651, 25652, 25653, 25654, 25655, 25656, 25657, 25658, 25659, 25660, 25661 Extra Details: protein stability; DCp; beta-turns; denatured state ensemble; thermophiles; urea denaturation; thermal denaturation

Submission Details

ID: oocd76v93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:55 p.m.

Version: 1

Publication Details
Fu H;Grimsley G;Scholtz JM;Pace CN,Protein Sci. (2010) Increasing protein stability: importance of DeltaC(p) and the denatured state. PMID:20340133
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