Engineering stabilising β-sheet interactions into a conformationally flexible region of the folding transition state of ubiquitin

被引:11
作者
Bofill, R [1 ]
Searle, MS [1 ]
机构
[1] Univ Nottingham, Ctr Biomol Sci, Nottingham NG7 2RD, England
关键词
protein folding; protein engineering; ubiquitin; beta-hairpin stabilisation; non-native interactions;
D O I
10.1016/j.jmb.2005.08.044
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
Protein engineering studies suggest that the transition state for the folding of ubiquitin is highly polarised towards the N-terminal part of the sequence and involves a nucleus of residues within the beta-hairpin (residues 1-17) and main alpha-helix (residues 23-34). In contrast, the observation of small phi-values for residues in the C-terminal portion of the sequence (residues 35-76), coupled with a folding topology that results in a much higher contact order, suggests that fast folding of ubiquitin is dependent upon configurational flexibility in the C-terminal part of the polypeptide chain to ensure passage down a relatively smooth folding funnel to the native state. We show that the introduction of a small mini-hairpin motif as an extension of the native 43-50 hairpin stabilises local interactions in the C-terminal part of the sequence, resulting largely in a deceleration of the unfolding kinetics without perturbing the apparent two-state folding mechanism. However, a single-point Leu -> Phe substitution within the engineered hairpin sequence leads to the premature collapse of the denatured ensemble through the stabilisation of non-native interactions and the population of a compact intermediate. Non-linear effects in the kinetic data at low concentrations of denaturant suggest that the collapsed state, which is further stabilised in the presence of cosmotropic salts, may subsequently fold directly to the native state through a "triangular" reaction scheme involving internal rearrangement rather than unfolding and refolding. (c) 2005 Elsevier Ltd. All rights reserved.
引用
收藏
页码:373 / 384
页数:12
相关论文
共 61 条
[1]   SPECIFIC NUCLEUS AS THE TRANSITION-STATE FOR PROTEIN-FOLDING - EVIDENCE FROM THE LATTICE MODEL [J].
ABKEVICH, VI ;
GUTIN, AM ;
SHAKHNOVICH, EI .
BIOCHEMISTRY, 1994, 33 (33) :10026-10036
[2]   Matching theory and experiment in protein folding [J].
Alm, E ;
Baker, D .
CURRENT OPINION IN STRUCTURAL BIOLOGY, 1999, 9 (02) :189-196
[3]   Protein folding - Making a network of hydrophobic clusters [J].
Baldwin, RL .
SCIENCE, 2002, 295 (5560) :1657-1658
[4]   Diffusion-collision model study of misfolding in a four-helix bundle protein [J].
Beck, C ;
Siemens, X ;
Weaver, DL .
BIOPHYSICAL JOURNAL, 2001, 81 (06) :3105-3115
[5]   Extending the folding nucleus of ubiquitin with an independently folding β-hairpin finger:: Hurdles to rapid folding arising from the stabilisation of local interactions [J].
Bofill, R ;
Simpson, ER ;
Platt, GW ;
Crespo, MD ;
Searle, MS .
JOURNAL OF MOLECULAR BIOLOGY, 2005, 349 (01) :205-221
[6]   DESTABILIZING EFFECTS OF REPLACING A SURFACE LYSINE OF CYTOCHROME-C WITH AROMATIC-AMINO-ACIDS - IMPLICATIONS FOR THE DENATURED STATE [J].
BOWLER, BE ;
MAY, K ;
ZARAGOZA, T ;
YORK, P ;
DONG, AC ;
CAUGHEY, WS .
BIOCHEMISTRY, 1993, 32 (01) :183-190
[7]   Im7 folding mechanism: misfolding on a path to the native state [J].
Capaldi, AP ;
Kleanthous, C ;
Radford, SE .
NATURE STRUCTURAL BIOLOGY, 2002, 9 (03) :209-216
[8]  
Chiti F, 1999, NAT STRUCT BIOL, V6, P1005
[9]   The effects of disulfide bonds on the denatured state of barnase [J].
Clarke, J ;
Hounslow, AM ;
Bond, CJ ;
Fersht, AR ;
Daggett, V .
PROTEIN SCIENCE, 2000, 9 (12) :2394-2404
[10]  
Cordes MHJ, 1999, PROTEIN SCI, V8, P318