Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM.

中文翻译：

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钙调素在调节骨骼肌钙释放中的解析结构状态

钙调蛋白 (CaM) 被提议通过依赖于 RyR1 结合 CaM 的构象的机制来调节骨骼肌肌浆网 (SR) 钙释放通道（兰尼碱受体，RyR1 异构体）的活性。然而，在生理相关条件下，CaM 结构与 RyR 功能调节之间的相关性在很大程度上是未知的。在这里，我们使用时间分辨荧光共振能量转移 (TR-FRET) 来研究 CaM 中可能在 RyR1 调节中起作用的结构变化。我们用荧光探针共价标记了 CaM 的每个叶（N 和 C），并使用分子内 TR-FRET 来评估当 CaM 与 SR 膜中的 RyR1、纯化的 RyR1 或对应于 RyR 的 CaM 结合域的肽时的叶间距离(RyRp)。TR-FRET 解决了 CaM 的两个不同结构状态（构象）之间的平衡，每个结构状态都以叶间距离和高斯分布宽度（无序）为特征。在孤立的 CaM 中，在低 Ca2+ 下，CaM 的两种构象被解析，集中在 5 nm（封闭）和 7 nm（开放）。在高 Ca2+ 时，平衡向有利于开放构象的方向移动。在高 Ca2+ 下存在 RyRp 时，闭合构象转变为更紧凑的构象并且是主要成分。当 CaM 与全长 RyR1 结合时，无论是纯化的还是在 SR 膜中，都会获得截然不同的结果：1) 两种构象得到解析且更加有序，2) 开放状态是主要成分，3) Ca2+ 稳定了2 倍的封闭构象。我们得出结论，与 RyR1 结合的 CaM 的 Ca2+ 依赖性结构分布与与 RyRp 结合的 CaM 的结构分布不同。我们建议将 RyR1 的功能调整为结合 CaM 的 Ca2+ 依赖性结构动力学。