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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion
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Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

机译：通过NMR弛豫分散法测量蛋白质的解折叠/折叠动力学和隐藏中间体的结构特征

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摘要

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use ~(15)N,~1H~N, and ~(13)CO NMR R_2 relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R_2 dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75-4.95 and determined to be an on-pathway intermediate (11) in a linear three-state unfolding scheme (N →← 11 →← MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N → 11 (36 s~(-1)) and MG → 11 (26 s~(-1)), respectively. Differences in chemical shift between N and 11 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R_2 dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N→ MG transition. The experiments also identify regions of energetic frustration that "crack" during unfolding and impede the refolding process.
机译：对蛋白质功能和功能障碍的详细了解取决于表征瞬时填充状态及其之间过渡的能力。在这里，我们使用〜（15）N，〜1H〜N和〜（13）CO NMR R_2弛豫分散液来研究天然载脂蛋白的自发解折叠和重折叠事件。 pH值高于5.0时，分散作用主要由涉及F螺旋区波动的过程决定，而这在NMR光谱中是看不见的。对于天然（N）结构中F-螺旋区域接触的残基，R_2分散度的测量揭示了由螺旋F的局部展开和与蛋白核心的对接而形成的瞬态。在pH 4.75-4.95处检测到类似状态，并被确定为线性三态展开方案（N→←11→←MG）的途中中间体（11），导致瞬时填充的熔融小球（MG）状态。展开和重新折叠的最慢步骤分别是N→11（36 s〜（-1））和MG→11（26 s〜（-1））。 N和11之间的化学位移差异很小，除了与螺旋F相邻的区域外，表明它们的核心结构相似。由R_2分散获得的N和MG状态之间的化学位移变化表明，瞬态MG状态在结构上类似于先前在高温和低pH下观察到的平衡MG。 MG状态化学位移的分析显示了残留的螺旋结构在过渡中间体中的位置，并确定了在N→MG过渡过程中展开或重排为非天然结构的区域。实验还确定了在展开过程中“破裂”并阻碍重新折叠过程的高能挫折区域。

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