Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange

被引:46
作者
Walkenhorst, WF
Edwards, JA
Markley, JL
Roder, H
机构
[1] Loyola Univ, Dept Chem, New Orleans, LA 70118 USA
[2] Fox Chase Canc Ctr, Inst Canc Res, Philadelphia, PA 19111 USA
[3] Univ Wisconsin, Dept Biochem, Madison, WI 53706 USA
[4] Univ Penn, Dept Biochem & Biophys, Philadelphia, PA 19104 USA
关键词
staphylococcal nuclease; pulse labeling; hydrogen exchange; NMR; protein folding;
D O I
10.1110/ps.ps.28202
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
Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly N-15-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the beta -barrel and to a lesser degree in the alpha -helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a beta -hairpin formed by strands 2 and 3 of the beta -barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the beta -sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.
引用
收藏
页码:82 / 91
页数:10
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