Molecular biophysics of motility in cytoskeletal motor proteins

本项目将研究肌动蛋白和肌球蛋白这两种蛋白质的结构和功能，这两种蛋白质属于被指定为“分子马达”的蛋白质类别。这些酶利用细胞中最常见的储能分子之一三磷酸腺苷的水解所产生的能量来驱动肌肉收缩、细胞内运输、细胞运动和细胞分裂。该项目的目标是了解这些蛋白质如何产生运动来执行细胞工作。这个项目将利用一种“金属拯救”技术，通过改变作为蛋白质必要组成部分的金属离子的比例来控制和调节马达蛋白质的活性。这项创新技术将有助于研究人员可逆地控制多种不同酶的活性。该项目的教育目标将填补当地教育系统的一个空白，以促进下一代科学家的科学教学和学习。这个项目的研究目标是利用生化、结构、生物物理和高等数学分析来确定非常规动蛋白和肌球蛋白马达蛋白的核苷酸水解机制。肌球蛋白和肌动蛋白超家族马达是细胞骨架细丝刺激的ATPase，它们在活性部位共享结构基序，直接与核苷酸和二价金属辅因子相互作用，典型的是镁(II)。金属拯救策略将被用来控制肌动蛋白和肌球蛋白的酶活性和运动性，并利用镁(II)和锰(II)对丝氨酸或半胱氨酸残基的不同亲和力。具体地说，对蛋白质-金属相互作用的操纵将提供一种直接的和实验上可逆的策略来调节Switch-1闭合，从而调节其细丝上的运动。确定了酿酒酵母野生型和金属抢救型Kinesin-5马达的ATPase机制，确定了盘基网柄菌野生型和金属挽救突变体的肌球蛋白-II马达的变构机械力化学。该项目将开发一种生物物理工具，以可逆地控制分子马达或其他P-环NTPase的活性。该项目得到了生物科学局分子和细胞生物科学司分子生物物理组的支持。

This project will investigate the structure and function of two proteins, kinesin and myosin, which belong to category of proteins that are designated as 'molecular motors'. These enzymes use the energy derived from hydrolysis of one of the most common energy storage molecules in cells, adenosine triphosphate, to drive muscle contraction, intracellular transport, cell motility, and cell division. The goal of the project is to understand how these proteins generate motion to perform cellular work. This project will utilize a 'metal-rescue' technology for controlling and modulating the activity of the motor protein by varying the ratio of metal ions that are necessary components for the proteins. This innovative technology will be useful for researchers to reversibly control the activity of multiple different enzymes. The educational aims of this project will fill a gap in the local education system to promote robust science teaching and learning for the next generation of scientists. Underrepresented minorities at the high school level will be educated and trained in STEM research.The research objective of this project is to define the nucleotide hydrolysis mechanisms of unconventional kinesin and myosin motor proteins using biochemical, structural, biophysical, and advanced mathematical analysis. Myosin and kinesin superfamily motors are cytoskeletal filament-stimulated ATPases that share structural motifs in their active sites which directly interact with the nucleotide and divalent metal cofactor, typically Mg(II). A 'metal-rescue' strategy will be used to control the enzymatic activity and motility of kinesins and myosins and by taking advantage of the differential affinities of Mg(II) and Mn(II) for serine or cysteine residues. Specifically, manipulation of the protein-metal interaction will provide a direct and experimentally reversible strategy to modulate switch-1 closure and, thus, motor motility upon its filament. The ATPase mechanism of wild type and the metal-rescue mutant kinesin-5 motors from Saccharomyces cerevisiae will be defined and the allosteric mechanochemistry of the wild type and metal-rescue mutant of myosin-II motors from Dictyostelium discoideum will be determined. This project will develop a biophysical tool to reversibly control the activity of molecular motors or other P-loop NTPases. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.