HA-detected experiments for the backbone assignment of intrinsically disordered proteins

被引:44
作者
Mantylahti, Sampo [1 ]
Aitio, Olli [1 ]
Hellman, Maarit [1 ]
Permi, Perttu [1 ]
机构
[1] Univ Helsinki, Program Struct Biol & Biophys, Inst Biotechnol, NMR Lab, FIN-00014 Helsinki, Finland
基金
芬兰科学院;
关键词
Assignment; EspF(U); H(CA)CON; iH(CA)NCO; Intrinsically unfolded proteins; TRIPLE-RESONANCE NMR; ISOTOPICALLY ENRICHED PROTEINS; PULSED-FIELD GRADIENTS; CHEMICAL-SHIFT; PROJECTION-RECONSTRUCTION; SEQUENTIAL ASSIGNMENT; UNSTRUCTURED PROTEINS; HYDROGEN-EXCHANGE; FOURIER-TRANSFORM; LABELED PROTEINS;
D O I
10.1007/s10858-010-9421-0
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
We propose a new alpha proton detection based approach for the sequential assignment of natively unfolded proteins. The proposed protocol superimposes on following features: HA-detection (1) enables assignment of natively unfolded proteins at any pH, i.e., it is not sensitive to rapid chemical exchange undergoing in natively unfolded proteins even at moderately high pH. (2) It allows straightforward assignment of proline-rich polypeptides without additional proline-customized experiments. (3) It offers more streamlined and less ambiguous assignment based on solely intraresidual (15)N(i)-(13)C'(i)-H(alpha)(i) (or (15)N(i)-(13)C(alpha)(i)-H(alpha)(i)) and sequential (15)N(i + 1)-(13)C'(i)-H(alpha)(i) (or (15)N(i + 1)-(13)C(alpha)(i)-H(alpha)(i)) correlation experiments together with efficient use of chemical shifts of (15)N and (13)C' nuclei, which show smaller dependence on residue type. We have tested the proposed protocol on two proteins, small globular 56-residue GB1, and highly disordered, proline-rich 47-residue fifth repeat of EspF(U). Using the proposed approach, we were able to assign 90% of (1)H(alpha), (13)C(alpha), (13)C', (15)N chemical shifts in EspF(U). We reckon that the HA-detection based strategy will be very useful in the assignment of natively unfolded proline-rich proteins or polypeptide chains.
引用
收藏
页码:171 / 181
页数:11
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