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
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:55 p.m.
|Number of data points||101|
|Proteins||Guanyl-specific ribonuclease Sa ; RIBONUCLEASE|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: dCp temp:60.0 C ; Experimental Assay: ddG ; Experimental Assay: dCp ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: ddG_H2O ; Derived Quantity: ddG_H2O ; Derived Quantity: dTm|
|Libraries||Mutations for sequence DVSGTVCLSALPPEATDTLNLIASDGPFPYSQDGVVFQNRESVLPTQSYGYYHEYTVITPGARTRGTRRIITGEATQEDYYTGDHYATFSLIDQTC|