Bergerac-SH3:: "Frustation" induced by stabilizing the folding nucleus

被引:27
作者
Viguera, AR
Serrano, L
机构
[1] EMBL, D-69117 Heidelberg, Germany
[2] CSIC, Unidad Biofis, Bilbao 48080, Spain
关键词
SH3; domain; folding kinetics; protein stability; beta-hairpin; folding transition state;
D O I
10.1006/jmbi.2001.4738
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The influence of an inserted exogenous independent folding element on the thermodynamics and folding properties of SH3 domain from alpha -spectrin has been investigated by creating a fused form between this small all-beta domain and a stable beta -hairpin (BH19). NMR analysis of synthetic peptides shows that insertion of BH19 nucleates formation of the original natural beta -hairpin (distal loop) that is part of the SH3 folding nucleus. The resulting protein (Bergerac-SHH) is more stable, folds faster and contains an elongated hairpin protruding from the globular domain as determined by 2D-NMR. "Protein engineering" analysis of the inserted region shows that it is folded in the transition state. Interestingly, stabilisation by insertion of the distal loop region results in the appearance of a compact intermediate revealed by a curved chevron plot at low denaturant concentration. This effect is eliminated at low salt concentrations by a single mutation of a hydrophobic residue within BH19 sequence, which is most probably involved in non-native interactions. Local stabilisation by enlargement and reinforcement of the folding nucleus, global compaction by the addition of salt and non-native interactions are shown to contribute to the observed deviation from the two-state behaviour. (C) 2001 Academic Press.
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页码:357 / 371
页数:15
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