Conformational strain in the hydrophobic core and its implications for protein folding and design.
We have designed de novo 13 divergent spectrin SH3 core sequences to determine their folding properties. Kinetic analysis of the variants with stability similar to that of the wild type protein shows accelerated unfolding and refolding rates compatible with a preferential stabilization of the transition state. This is most likely caused by conformational strain in the native state, as deletion of a methyl group (Ile-->Val) leads to deceleration in unfolding and increased stability (up to 2 kcal x mol(-1)). Several of these Ile-->Val mutants have negative phi(-U) values, indicating that some noncanonical phi(-U) values might result from conformational strain. Thus, producing a stable protein does not necessarily mean that the design process has been entirely successful. Strained interactions could have been introduced, and a reduction in the buried volume could result in a large increase in stability and a reduction in unfolding rates.
Ventura
Salvador
S
European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Vega
Maria Cristina
MC
Lacroix
Emmanuel
E
Angrand
Isabelle
I
Spagnolo
Laura
L
Serrano
Luis
L
D000465:Algorithms; D019943:Amino Acid Substitution; D000818:Animals; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010802:Phylogeny; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D017510:Protein Folding; D013049:Spectrin / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D018909:src Homology Domains / Q000235:genetics
D013049:Spectrin; D014508:Urea