Native and non-native secondary structure and dynamics in the pH 4 intermediate of apomyoglobin
Native and non-native secondary structure and dynamics in the pH 4 intermediate of apomyoglobin
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DOI：
10.1021/bi992545f
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发表时间：
2000-03-21
期刊：
影响因子：
2.9
通讯作者：
Wright, PE
中科院分区：
文献类型：
--
作者：
Eliezer, D;Chung, J;Wright, PE
在pH 4的部分折叠状态的脱辅基肌红蛋白代表了一个很好的模型毛皮一个强制性的动力学折叠中间体。这种中间态的结构和动力学已经被广泛地使用NMR光谱学研究。二级化学位移，H-1-H-1 NOE，和酰胺质子温度系数已被用来探测在中间状态的残留结构，和NMR弛豫参数T-1和T-2和（H-1）-N-15 NOE已被分析使用光谱密度相关的多肽链的运动与这些结构的观察。大量的螺旋结构保持在pH 4状态，由C-13（alpha）、（CO）-C-13、H-1（alpha）和C-13（beta）核的次级化学位移指示，并且该螺旋结构的边界由H-1-H-1 NOE的位置确认。氢键在结构化区域主要是nativelike根据酰胺质子化学位移和它们的温度依赖性。的A，G，和H螺旋段和C-末端部分的B螺旋的位置是类似的那些在天然脱辅基肌红蛋白，与早期，完全保护这些螺旋中的残基的酰胺在淬灭流实验。这些结果证实了apoMb在pH 4时的平衡形式与猝灭流实验中短时间内观察到的动力学中间体的相似性。与蛋白质的其余部分相比，该结构化核心的灵活性严重缩减，如NMR弛豫参数分析所示。具有相对高的J（0）值和低的J（750）值的区域与A、B、G和H螺旋很好地对应，这表明在序列的这些区域中纳秒时间尺度的主链波动受到限制。蛋白质的其他部分显示出更大的灵活性和更低的二级化学位移。然而，有几个区域显示出螺旋结构开始的证据，包括包含C螺旋-CD环的延伸、D和E螺旋的边界以及E螺旋的C末端的一半。这些区域在pH 4状态下显然不是结构良好的，不像A、B、G和H螺旋，它们形成类似天然的结构核心。然而，这种结构化核心的接近性最有可能影响B和F螺旋之间的区域，从而诱导至少瞬时的螺旋结构。
The partly folded state of apomyoglobin at pH 4 represents an excellent model fur an obligatory kinetic folding intermediate. The structure and dynamics of this intermediate state have been extensively examined using NMR spectroscopy. Secondary chemical shifts, H-1-H-1 NOEs, and amide proton temperature coefficients have been used to probe residual structure in the intermediate state, and NMR relaxation parameters T-1 and T-2 and (H-1)-N-15 NOE have been analyzed using spectral densities to correlate motion of the polypeptide chain with these structural observations. A significant amount of helical structure remains in the pH 4 state, indicated by the secondary chemical shifts of the C-13(alpha), (CO)-C-13, H-1(alpha), and C-13(beta) nuclei, and the boundaries of this helical structure are confirmed by the locations of H-1-H-1 NOEs. Hydrogen bonding in the structured regions is predominantly nativelike according to the amide proton chemical shifts and their temperature dependence. The locations of the A, G, and H helix segments and the C-terminal part of the B helix are similar to those in native apomyoglobin, consistent with the early, complete protection of the amides of residues in these helices in quench-flow experiments. These results confirm the similarity of the equilibrium form of apoMb at pH 4 and the kinetic intermediate observed at short times in the quench-flow experiment. Flexibility in this structured core is severely curtailed compared with the remainder of the protein, as indicated by the analysis of the NMR relaxation parameters. Regions with relatively high values of J(0) and low values of J(750) correspond well with the A, B, G, and H helices, an indication that nanosecond time scale backbone fluctuations in these regions of the sequence are restricted. Other parts of the protein show much greater flexibility and much reduced secondary chemical shifts. Nevertheless, several regions show evidence of the beginnings of helical structure, including stretches encompassing the C helix-CD loop, the boundary of the D and E helices, and the C-terminal half of the E helix. These regions are clearly not well-structured in the pH 4 state, unlike the A, B, G, and H helices, which form a nativelike structured core. However, the proximity of this structured core most likely influences the region between the B and F helices, inducing at least transient helical structure.