HA-detected experiments for the backbone assignment of intrinsically disordered proteins
HA-detected experiments for the backbone assignment of intrinsically disordered proteins
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DOI：
10.1007/s10858-010-9421-0
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发表时间：
2010-07-01
影响因子：
2.7
通讯作者：
Permi, Perttu
中科院分区：
文献类型：
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作者：
Mantylahti, Sampo;Aitio, Olli;Permi, Perttu
我们提出了一种新的基于α质子检测的天然未折叠蛋白序列分配方法。提出的方案叠加在以下特征上：ha检测(1)能够在任何pH下分配天然未折叠蛋白，即即使在中等高pH下，它对天然未折叠蛋白中发生的快速化学交换也不敏感。(2)它允许直接分配富含脯氨酸的多肽，而无需额外的脯氨酸定制实验。(3)它提供了更精简和更少歧义的分配，仅基于残余内（15）N(i)-(13)C'(i)- h (α)(i)(或（15）N(i)-(13)C(α)(i)- h （α)(i)）和顺序（15）N(i + 1)-(13)C‘(i)- h (α)(i))(或（15）N(i + 1)-(13)C(α)(i)- h （α)(i)）相关实验，并有效利用（15）N和（13）C’原子核的化学位移，对残基类型的依赖性较小。我们已经在两种蛋白质上测试了所提出的方案，一种是小球状56-残基GB1，另一种是高度无序的富含脯氨酸的47-残基EspF(U)的第五次重复。利用所提出的方法，我们能够在EspF(U)中分配90%的(1)H(α), (13)C(α), (13)C', (15)N化学位移。我们认为基于ha检测的策略将在天然展开的富含脯氨酸的蛋白质或多肽链的分配中非常有用。
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.