Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability.


Abstract

We have chosen two members of the microbial RNase family, barnase and binase, which have 85% identity (17 substitutions and 1 deletion) and almost identical three-dimensional structure, to study the evolution of protein stability. The 17 residues that differ are scattered throughout the molecule. Each of the 17 differing residues has been mutated independently and the effect on protein stability analysed. Each point mutation has an effect on protein stability that ranges from +1.1 to -1.1 kcal mol-1. These changes in energy are additive. There is no clear correlation between the type of mutation and the effect on protein stability. A multiple mutant having six of the single mutations that increase the stability of barnase is 3.3 kcal mol-1 more stable than wild type and has the same activity. There could be selective pressure to maintain proteins at a certain stability and, consequently, mutations that decrease stability tend to be counterbalanced by stabilizing mutations. Alternatively, there could simply be pressure to maintain stability above a certain level, and any further increases in stability need not be maintained during evolution. These results suggest a simple way to improve the stability of proteins: choose two homologous proteins that have high similarity, mutate individually all of the residues that differ between the two, and combine the mutations that increase the stability in a multiple mutant. Study holds ProTherm entries: 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 2226, 2227 Extra Details: dG and ddG were measured in the presence of [urea]50% protein stability; co-operativity; protein evolution; barnase

Submission Details

ID: FYodeXWJ4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Serrano L;Day AG;Fersht AR,J. Mol. Biol. (1993) Step-wise mutation of barnase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. PMID:8377205
Additional Information

Sequence Assay Result Units