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
1AY7 1997-11-14T00:00:00+0000 1.7 RIBONUCLEASE SA COMPLEX WITH BARSTAR
1BOX 1998-08-07T00:00:00+0000 1.6 N39S MUTANT OF RNASE SA FROM STREPTOMYCES AUREOFACIENS
1C54 1999-10-22T00:00:00+0000 0 SOLUTION STRUCTURE OF RIBONUCLEASE SA
1GMP 1992-10-01T00:00:00+0000 1.7 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1GMQ 1992-10-01T00:00:00+0000 1.8 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1GMR 1992-10-01T00:00:00+0000 1.77 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1I70 2001-03-07T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF RNASE SA Y86F MUTANT
1I8V 2001-03-16T00:00:00+0000 1.25 CRYSTAL STRUCTURE OF RNASE SA Y80F MUTANT
1LNI 2002-05-03T00:00:00+0000 1.0 CRYSTAL STRUCTURE ANALYSIS OF A RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS AT ATOMIC RESOLUTION (1.0 A)
1RGE 1995-06-05T00:00:00+0000 1.15 HYDROLASE, GUANYLORIBONUCLEASE

Relevant UniProtKB Entries

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