Probing slowly exchanging protein systems via 13Cα-CEST: monitoring folding of the Im7 protein

被引:21
作者
Hansen, Alexandar L. [1 ,2 ,3 ]
Bouvignies, Guillaume [1 ,2 ,3 ]
Kay, Lewis E. [1 ,2 ,3 ,4 ]
机构
[1] Univ Toronto, Dept Mol Genet, Toronto, ON M5S 1A8, Canada
[2] Univ Toronto, Dept Biochem, Toronto, ON M5S 1A8, Canada
[3] Univ Toronto, Dept Chem, Toronto, ON M5S 1A8, Canada
[4] Hosp Sick Children, Program Mol Struct & Funct, Toronto, ON M5G 1X8, Canada
基金
加拿大自然科学与工程研究理事会; 美国国家科学基金会; 加拿大健康研究院;
关键词
Protein dynamics; Slow chemical exchange; Chemical exchange saturation transfer (CEST); Protein folding; Im7; DISPERSION NMR-SPECTROSCOPY; TIME-SCALE DYNAMICS; CHEMICAL-SHIFTS; C-ALPHA; ACCURATE MEASUREMENT; MOLECULAR-DYNAMICS; PULSE SEQUENCES; RELAXATION; STATES; INTERMEDIATE;
D O I
10.1007/s10858-013-9711-4
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
A C-13(alpha) chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an 'observable' ground state and one or more 'invisible' excited states. Here a labeling strategy whereby [2-C-13]-glucose is the sole carbon source is exploited, producing proteins with C-13 at the C-alpha position, while the majority of residues remain unlabeled at CO or C-beta. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state C-13(alpha) chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.
引用
收藏
页码:279 / 289
页数:11
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