Department of Chemistry and Center for Photochemical Science, Bowling Green State University, Bowling Green, Ohio 43403, USA.
J Chem Phys. 2022 Feb 7;156(5):055102. doi: 10.1063/5.0074277.
Conformational dynamics play a crucial role in protein functions. A molecular-level understanding of the conformational transition dynamics of proteins is fundamental for studying protein functions. Here, we report a study of real-time conformational dynamic interaction between calcium-activated calmodulin (CaM) and C28W peptide using single-molecule fluorescence resonance energy transfer (FRET) spectroscopy and imaging. Plasma membrane Ca-ATPase protein interacts with CaM by its peptide segment that contains 28 amino acids (C28W). The interaction between CaM and the Ca-ATPase is essential for cell signaling. However, details about its dynamic interaction are still not clear. In our current study, we used Cyanine3 labeled CaM (N-domain) and Dylight 649 labeled C28W peptide (N-domain) to study the conformational dynamics during their interaction. In this study, the FRET can be measured when the CaM-C28W complex is formed and only be observed when such a complex is formed. By using single-molecule FRET efficiency trajectory and unique statistical approaches, we were able to observe multiple binding steps with detailed dynamic features of loosely bound and tightly bound state fluctuations. The C-domain of CaM tends to bind with C28W first with a higher affinity, followed by the binding of the CaM N-domain. Due to the comparatively high flexibility and low affinity of the N-domain and the presence of multiple anchor hydrophobic residues on the peptide, the N-domain binding may switch between selective and non-selective binding states, while the C-domain remains strongly bound with C28W. The results provide a mechanistic understanding of the CaM signaling interaction and activation of the Ca-ATPase through multiple-state binding to the C28W. The new single-molecule spectroscopic analyses demonstrated in this work can be applied for broad studies of protein functional conformation fluctuation and protein-protein interaction dynamics.
构象动态在蛋白质功能中起着至关重要的作用。从分子水平上了解蛋白质构象转变动力学对于研究蛋白质功能至关重要。在这里，我们使用单分子荧光共振能量转移（FRET）光谱和成像技术报告了钙调蛋白（CaM）与 C28W 肽之间实时构象动态相互作用的研究。质膜 Ca-ATPase 蛋白通过含有 28 个氨基酸的肽段与 CaM 相互作用（C28W）。CaM 与 Ca-ATPase 的相互作用对于细胞信号转导至关重要。然而，其动态相互作用的细节仍不清楚。在我们目前的研究中，我们使用 Cy3 标记的 CaM（N 结构域）和 Dylight 649 标记的 C28W 肽（N 结构域）来研究它们相互作用过程中的构象动力学。在这项研究中，只有当 CaM-C28W 复合物形成时才能测量 FRET，并且只有当形成这样的复合物时才能观察到 FRET。通过使用单分子 FRET 效率轨迹和独特的统计方法，我们能够观察到具有松散结合和紧密结合状态波动的详细动态特征的多个结合步骤。CaM 的 C 结构域首先与 C28W 以更高的亲和力结合，然后是 CaM N 结构域的结合。由于 N 结构域的相对较高的灵活性和低亲和力以及肽上存在多个锚定疏水性残基，N 结构域的结合可能在选择性和非选择性结合状态之间切换，而 C 结构域与 C28W 保持强烈结合。该结果提供了 CaM 信号转导相互作用和通过多态结合对 Ca-ATPase 的激活的机制理解。本工作中展示的新的单分子光谱分析可以广泛应用于蛋白质功能构象波动和蛋白质-蛋白质相互作用动力学的研究。
