標題: Compact dimension of denatured states of staphylococcal nuclease
作者: Chow, C. -Y.
Wu, Ming-Chya
Fang, Huey-Jen
Hu, Chin-Kun
Chen, Hueih-Min
Tsong, Tian-Yow
交大名義發表
National Chiao Tung University
關鍵字: staphylococcal nuclease;differential scanning microcalorimetry;fluorescence energy transfer;denatured state;folding kinetics
公開日期: 15-Aug-2008
摘要: Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5 '-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 angstrom while it is 20.4 angstrom for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model. No reversible arrow D-1 reversible arrow D-2 reversible arrow D-3 (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.
URI: http://dx.doi.org/10.1002/prot.21985
http://hdl.handle.net/11536/8462
ISSN: 0887-3585
DOI: 10.1002/prot.21985
期刊: PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
Volume: 72
Issue: 3
起始頁: 901
結束頁: 909
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