Department of Biochemistry, Biophysics and Molecular Biology, University of Minnesota, Minneapolis, MN 55455, USA.
Int J Mol Sci. 2018 Sep 5;19(9):2628. doi: 10.3390/ijms19092628.
Actin-myosin interactions play crucial roles in the generation of cellular force and movement. The molecular mechanism involves structural transitions at the interface between actin and myosin's catalytic domain, and within myosin's light chain domain, which contains binding sites for essential (ELC) and regulatory light chains (RLC). High-resolution crystal structures of isolated actin and myosin, along with cryo-electron micrographs of actin-myosin complexes, have been used to construct detailed structural models for actin-myosin interactions. However, these methods are limited by disorder, particularly within the light chain domain, and they do not capture the dynamics within this complex under physiological conditions in solution. Here we highlight the contributions of site-directed fluorescent probes and time-resolved fluorescence resonance energy transfer (TR-FRET) in understanding the structural dynamics of the actin-myosin complex in solution. A donor fluorescent probe on actin and an acceptor fluorescent probe on myosin, together with high performance TR-FRET, directly resolves structural states in the bound actin-myosin complex during its interaction with adenosine triphosphate (ATP). Results from these studies have profound implications for understanding the contractile function of actomyosin and establish the feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.
肌动球蛋白相互作用在细胞力和运动的产生中起着至关重要的作用。分子机制涉及肌动蛋白和肌球蛋白催化结构域之间界面以及肌球蛋白轻链结构域的结构转变，后者包含结合必需（ELC）和调节轻链（RLC）的结合位点。已使用分离的肌动蛋白和肌球蛋白的高分辨率晶体结构以及肌动球蛋白复合物的低温电子显微镜图来构建肌动球蛋白相互作用的详细结构模型。然而，这些方法受到无序的限制，特别是在轻链结构域内，并且它们不能在溶液中生理条件下捕获该复合物内的动力学。在这里，我们强调了定点荧光探针和时间分辨荧光共振能量转移（TR-FRET）在理解溶液中肌动球蛋白复合物结构动力学中的贡献。肌动蛋白上的供体荧光探针和肌球蛋白上的受体荧光探针，以及高性能 TR-FRET，可直接解析与三磷酸腺苷（ATP）相互作用期间结合的肌动球蛋白复合物中的结构状态。这些研究的结果对理解肌动球蛋白的收缩功能具有深远的意义，并为发现肌动球蛋白相互作用的变构调节剂奠定了基础，最终目标是开发肌肉疾病的治疗方法。
