Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland.
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland.
Biophys J. 2020 Mar 10;118(5):1119-1128. doi: 10.1016/j.bpj.2020.01.003. Epub 2020 Jan 15.
Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates the exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigated the effect, using NMR spectroscopy, of substituting key charged Arg, Lys, Glu, and Asp residues by Gly or His. Results indicate that such mutations result in the transient breaking of the helix at the site of mutation but with noticeable impact on amide hydrogen exchange rates extending as far as ±2 helical turns, pointing to a substantial degree of cooperativity in SAH stability. Whereas a single Gly substitution caused transient breaks ∼20% of the time, two consecutive Gly substitutions break the helix ∼65% of the time. NMR relaxation measurements indicate that the exchange rate between an intact and a broken helix is fast (>300,000 s) and that for the wild-type sequence, the finite persistence length is dominated by thermal fluctuations of backbone torsion angles and H-bond lengths, not by transient helix breaking. The double mutation D27H/E28H causes a pH-dependent fraction of helix disruption, in which the helix breakage increases from 26% at pH 7.5 to 53% at pH 5.5. The ability to modulate helical integrity by pH may enable incorporation of externally tunable dynamic components in the design of molecular machines.
带高电荷的、单一α螺旋（SAH）结构域富含精氨酸（Arg）、赖氨酸（Lys）和谷氨酸（Glu）残基。其动态盐桥配对产生了这些螺旋棒的特殊刚性，肌球蛋白 VI SAH 结构域的持久长度超过 200Å。为了调节螺旋结构的刚性，我们使用 NMR 光谱研究了通过取代关键的带电荷的 Arg、Lys、Glu 和 Asp 残基为 Gly 或 His 对其的影响。结果表明，这种突变导致在突变部位的螺旋瞬时断裂，但对酰胺氢交换率有显著影响，延伸至±2 个螺旋圈，表明 SAH 稳定性存在相当大的协同性。虽然单个 Gly 取代导致瞬时断裂的时间约为 20%，但两个连续的 Gly 取代导致螺旋断裂的时间约为 65%。NMR 弛豫测量表明，完整和断裂螺旋之间的交换速率非常快（>300,000 s），对于野生型序列，有限的持久长度主要由主链扭转角和氢键长度的热波动决定，而不是由瞬时螺旋断裂决定。双突变 D27H/E28H 导致 pH 依赖性的螺旋破坏分数，其中螺旋断裂从 pH 7.5 时的 26%增加到 pH 5.5 时的 53%。通过 pH 调节螺旋完整性的能力可能使在分子机器的设计中可以纳入外部可调动态组件。
