Lee Jung Ho, Ying Jinfa, Bax Ad
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Phys Chem Chem Phys. 2016 Feb 17;18(8):5759-70. doi: 10.1039/c5cp04542h.
(3)JHNHα and (3)JC'C' couplings can be readily measured in isotopically enriched proteins and were shown to contain precise information on the backbone torsion angles, ϕ, sampled in disordered regions of proteins. However, quantitative interpretation of these couplings required the population of conformers with positive ϕ angles to be very small. Here, we demonstrate that this restriction can be removed by measurement of (3)JC'Hα values. Even though the functional forms of the (3)JC'Hα and (3)JHNHα Karplus equations are the same, large differences in their coefficients enable accurate determination of the fraction of time that positive ϕ angles are sampled. A four-dimensional triple resonance HACANH[C'] E.COSY experiment is introduced to simultaneously measure (3)JC'Hα and (3)JHNC' in the typically very congested spectra of disordered proteins. High resolution in these spectra is obtained by non-uniform sampling (in the 0.1-0.5% range). Application to the intrinsically disordered protein α-synuclein shows that while most residues have close-to-zero positive ϕ angle populations, up to 16% positive ϕ population is observed for Asn residues. Positive ϕ angle populations determined with the new approach agree closely with consensus values from protein coil libraries and prior analysis of a large set of other NMR parameters. The combination of (3)JHNC' and (3)JC'C' provides information about the amplitude of ϕ angle dynamics.
（3）JHNHα和（3）JC'C'耦合可以在同位素富集的蛋白质中轻松测量，并且已证明它们包含有关蛋白质无序区域中采样的主链扭转角ϕ的精确信息。然而，对这些耦合进行定量解释需要具有正ϕ角的构象异构体的数量非常少。在这里，我们证明通过测量（3）JC'Hα值可以消除这一限制。尽管（3）JC'Hα和（3）JHNHα Karplus方程的函数形式相同，但它们系数的巨大差异使得能够准确确定采样正ϕ角的时间分数。引入了一种四维三重共振HACANH[C'] E.COSY实验，以在通常非常拥挤的无序蛋白质光谱中同时测量（3）JC'Hα和（3）JHNC'。通过非均匀采样（在0.1 - 0.5%范围内）获得这些光谱中的高分辨率。应用于内在无序蛋白α-突触核蛋白表明，虽然大多数残基的正ϕ角数量接近零，但对于Asn残基观察到高达16%的正ϕ角数量。用新方法确定的正ϕ角数量与蛋白质卷曲库的共识值以及对大量其他NMR参数的先前分析密切一致。（3）JHNC'和（3）JC'C'的组合提供了有关ϕ角动力学幅度的信息。
