The folding pathway of barnase: The rate-limiting transition state and a hidden intermediate under native conditions

被引:39
作者
Vu, ND [1 ]
Feng, HQ [1 ]
Bai, YW [1 ]
机构
[1] NCI, Biochem Lab, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1021/bi0362267
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 nature of the rate-limiting transition state at zero denaturant (TS1) and whether there are hidden intermediates are the two major unsolved problems in defining the folding pathway of barnase. In earlier studies, it was shown that TS1 has small phi values throughout the structure of the protein, suggesting that the transition state has either a defined partially folded secondary structure with all side chains significantly exposed or numerous different partially unfolded structures with similar stability. To distinguish the two possibilities, we studied the effect of Gly mutations on the folding rate of barnase to investigate the secondary structure formation in the transition state. Two mutations in the same region of a beta-strand decreased the folding rate by 20- and 50-fold, respectively, suggesting that the secondary structures in this region are dominantly formed in the rate-limiting transition state. We also performed native-state hydrogen exchange experiments on barnase at pD 5.0 and 25 degreesC and identified a partially unfolded state. The structure of the intermediate was investigated using protein engineering and NMR. The results suggest that the intermediate has an omega loop unfolded. This intermediate is more folded than the rate-limiting transition state previously characterized at high denaturant concentrations (TS2). Therefore, it exists after TS2 in folding. Consistent with this conclusion, the intermediate folds with the same rate and denaturant dependence as the wild-type protein, but unfolds faster with less dependence on the denaturant concentration. These and other results in the literature suggest that barnase folds through partially unfolded intermediates that exist after the rate-limiting step. Such folding behavior is similar to those of cytochrome c and Rdapocyt b(562). Together, we suggest that other small apparently two-state proteins may also fold through hidden intermediates.
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页码:3346 / 3356
页数:11
相关论文
共 56 条
[1]   A COMPARISON OF THE PH, UREA, AND TEMPERATURE-DENATURED STATES OF BARNASE BY HETERONUCLEAR NMR - IMPLICATIONS FOR THE INITIATION OF PROTEIN-FOLDING [J].
ARCUS, VL ;
VUILLEUMIER, S ;
FREUND, SMV ;
BYCROFT, M ;
FERSHT, AR .
JOURNAL OF MOLECULAR BIOLOGY, 1995, 254 (02) :305-321
[2]   PRIMARY STRUCTURE EFFECTS ON PEPTIDE GROUP HYDROGEN-EXCHANGE [J].
BAI, YW ;
MILNE, JS ;
MAYNE, L ;
ENGLANDER, SW .
PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1993, 17 (01) :75-86
[3]   Hidden intermediates and levinthal paradox in the folding of small proteins [J].
Bai, YW .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, 305 (04) :785-788
[4]  
Bai YW, 1996, PROTEINS, V24, P145, DOI 10.1002/(SICI)1097-0134(199602)24:2<145::AID-PROT1>3.0.CO
[5]  
2-I
[6]   The problem was to find the problem [J].
Baldwin, RL .
PROTEIN SCIENCE, 1997, 6 (09) :2031-2034
[7]   The energy landscape of a fast-folding protein mapped by Ala->Gly substitutions [J].
Burton, RE ;
Huang, GS ;
Daugherty, MA ;
Calderone, TL ;
Oas, TG .
NATURE STRUCTURAL BIOLOGY, 1997, 4 (04) :305-310
[8]   DETECTION AND CHARACTERIZATION OF A FOLDING INTERMEDIATE IN BARNASE BY NMR [J].
BYCROFT, M ;
MATOUSCHEK, A ;
KELLIS, JT ;
SERRANO, L ;
FERSHT, AR .
NATURE, 1990, 346 (6283) :488-490
[9]   Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH [J].
Chamberlain, AK ;
Handel, TM ;
Marqusee, S .
NATURE STRUCTURAL BIOLOGY, 1996, 3 (09) :782-787
[10]   Lack of definable nucleation sites in the rate-limiting transition state of barnase under native conditions [J].
Chu, RA ;
Bai, YW .
JOURNAL OF MOLECULAR BIOLOGY, 2002, 315 (04) :759-770