Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy
Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy
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DOI：
10.1021/jacs.9b03116
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发表时间：
2019-06-05
影响因子：
15
通讯作者：
Bax, Ad
中科院分区：
文献类型：
--
作者：
Barnes, C. Ashley;Shen, Yang;Bax, Ad
虽然α -螺旋一直被认为是二级结构的重要组成部分，但它通常需要通过与蛋白质结构的其他部分的三级相互作用来稳定。高电荷的单α -螺旋（SAH）结构域，由高百分比（bbb75 %）的Arg， Lys和Glu残基组成，是这一规则的例外，但很难在结构上表征。我们的研究重点是肌球蛋白- vi的68个残基内侧尾结构域，发现它包含一个高度有序的α -螺旋结构，从glu6延伸到Lys-63。在丝状噬菌体培养基中测量的高氢交换保护因子（15-150），小（约4 Hz） (3)J（HNH α）偶联，以及近乎完美地适合其残余偶极偶联（rdc）的理想α -螺旋模型，支持该螺旋的高度规则性。值得注意的是，从它们得到的氢交换率是更加均匀的保护因子，这表明对于这些短暂断裂的螺旋，从氨基酸序列得到的本征交换率不是合适的参考值。N-15弛豫数据显示了非常高的旋转扩散各向异性（d平行于/ d垂直于接近7.6），与预测的这种长而接近直的α -螺旋的流体动力学行为一致。顺磁性镧系离子附着在其n端区域的螺旋排列显示，随着距离标记位点的增加，排列减少。在20摄氏度下，这种减少产生了224 +/- 10埃的持续长度的精确测量，支持了SAH螺旋的作用是作为肌球蛋白- vi杠杆臂的延伸的观点。
Although the alpha-helix has long been recognized as an all-important element of secondary structure, it generally requires stabilization by tertiary interactions with other parts of a protein's structure. Highly charged single alpha-helical (SAH) domains, consisting of a high percentage (>75%) of Arg, Lys, and Glu residues, are exceptions to this rule but have been difficult to characterize structurally. Our study focuses on the 68-residue medial tail domain of myosin-VI, which is found to contain a highly ordered alpha-helical structure extending from Glu-6 to Lys-63. High hydrogen exchange protection factors (15-150), small (ca. 4 Hz) (3)J(HNH alpha) couplings, and a near-perfect fit to an ideal model alpha-helix for its residual dipolar couplings (RDCs), measured in a filamentous phage medium, support the high regularity of this helix. Remarkably, the hydrogen exchange rates are far more homogeneous protection factors derived from them, suggesting that for these transiently broken helices the intrinsic exchange rates derived from the amino acid sequence are not appropriate reference values. N-15 relaxation data indicate a very high degree of rotational diffusion anisotropy (D-parallel to/D-perpendicular to approximate to 7.6), consistent with the hydrodynamic behavior predicted for such a long, nearly straight alpha-helix. Alignment of the helix by a paramagnetic lanthanide ion attached to its N-terminal region shows a decrease in alignment as the distance from the tagging site increases. This decrease yields a precise measure for the persistence length of 224 +/- 10 angstrom at 20 degrees C, supporting the idea that the role of the SAH helix is to act as an extension of the myosin-VI lever arm.