Conversion of two-state to multi-state folding kinetics on fusion of two protein foldons

被引:23
作者
Inaba, K [1 ]
Kobayashi, N [1 ]
Fersht, AR [1 ]
机构
[1] MRC Ctr, Cambridge Ctr Prot Engn, Cambridge CB2 2QH, England
基金
日本学术振兴会;
关键词
CI2; foldon; folding intermediate; protein design;
D O I
10.1006/jmbi.2000.4024
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
Chymotrypsin inhibitor 2 (CI2) is the archetypal single-foldon protein that folds in simple two-state kinetics without the accumulation of a folding intermediate. To model the effects of fusion of single foldons to give a multi-foldon protein, we engineered a "double-CI2" protein, in which another CI2 polypeptide was inserted into the loop region of the parent CI2. CD and HSQC spectra demonstrated that while the double-CI2 protein adopted two kinds of native conformations, CI2-like structure was almost preserved in both the domains of double-CI2. In the folding kinetic studies, double-CI2 exhibited a remarkable rollover of the observed folding rates at low denaturant concentrations, indicating that double-CI2 accumulated a kinetic folding intermediate. The different folding mechanisms between WT-CI2 and double-CI2 support the present view that protein size or number of domains is an important determinant for formation of folding intermediates. (C) 2000 Academic Press.
引用
收藏
页码:219 / 233
页数:15
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