NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions

被引:152
作者
Mok, YK
Kay, CM
Kay, LE
Forman-Kay, J
机构
[1] Hosp Sick Children, Struct Biol & Biochem Programme, Toronto, ON M5G 1X8, Canada
[2] Univ Toronto, Dept Biochem, Toronto, ON M5S 1A8, Canada
[3] Univ Alberta, Dept Biochem, Edmonton, AB T6G 2H7, Canada
[4] Univ Alberta, Prot Engn Network Ctr Excellence, Edmonton, AB T6G 2H7, Canada
[5] Univ Toronto, Dept Chem Biochem, Toronto, ON M5S 1A8, Canada
[6] Univ Toronto, Dept Mol & Med Genet, Toronto, ON M5S 1A8, Canada
[7] Univ Toronto, Prot Engn Network Ctr Excellence, Toronto, ON M5S 1A8, Canada
基金
英国医学研究理事会;
关键词
unfolded state; structure; NMR; NOE; deuteration;
D O I
10.1006/jmbi.1999.2769
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 N-terminal SH3 domain of drk (drkN SH3) is unstable, existing in equilibrium between a folded state (F-exch) and an unfolded state (U-exch) under non-denaturing buffer conditions. Using a N-15/H-2-Iabeled sample, long range amide NOEs can be observed in the U-exch state as a result of reduced relaxation, in some cases correlating protons over 40 residues apart. These long range NOEs disappear upon addition of 2 M guanidinium chloride, demonstrating that there are substantial differences between the U-exch and the guanidine denatured states. Calculations using the long range NOEs of the U-exch state yield highly compact structures having non-native turns and a non-native buried tryptophan residue. These structures agree with experimental stopped-flow fluorescence data and analytical ultracentrifugation results. Since protein stability depends on the structural and dynamic properties of both the folded and unfolded states, this study provides insights into the stability of the drkN SH3 domain. These results provide the first strong NOE-based evidence for compact unfolded states of proteins and suggest that some unfolded states under physiological conditions have specific interactions leading to compact structures. (C) 1999 Academic Press.
引用
收藏
页码:619 / 638
页数:20
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