MYOSIN DYNAMICS DURING THE CONTRACTILE CYCLE

该计划项目的主题是了解分子马达如何工作，特别是阐明肌球蛋白在收缩周期中发生的结构转变。为了解决这些问题，阐明肌球蛋白在收缩周期中发生的结构转变。为了解决这些问题，这四个项目结合了一系列最先进的技术，包括单分子机械测量、体外动力分析、荧光光谱分析、真核系统中的蛋白质表达以及蛋白质生物化学和表征。在项目1中，Berger博士将在溶液和单分子水平上使用光谱方法来确定杠杆臂在收缩周期中如何倾斜，并在单分子水平上确定杠杆臂在收缩周期中如何倾斜，以及裂隙是否打开和关闭以调节其与肌动蛋白的亲和力。华沙博士(项目2)将使用激光陷阱和全内反射显微镜来同时测量单个肌球蛋白分子及其活性部位的核苷酸(单分子荧光)或杠杆臂的取向(荧光偏振)。项目#3(Trybus博士)将使用一种结合生化和结构技术(冷冻电子显微镜、结晶学)的突变方法来破译肌球蛋白的两个头部如何协作和运动产生，肌球蛋白界面在PowerStroke过程中如何变化，以及杠杆臂的启动是否需要ATP水解。Lowey博士(项目#4)建议使用冷冻电子显微镜技术测试肌动球蛋白界面和杠杆臂中结构域移动的一般性。还将确定肌动蛋白和肌球蛋白的取向是否在单分子水平上影响工作中风。长期的目标是了解肌球蛋白的每个结构域如何对化学机械耦合做出贡献，以便有可能推导出一套分子马达的设计原则。

The theme of this Program Project is to understand how molecular motor work, and in particular to elucidate the structural transitions that occur in myosin during the contractile cycle. To address these elucidate the structural transitions that occur in myosin during the contractile cycle. To address these questions, the four projects bring together a wide array of state-of-the art techniques, including single molecular mechanical measurements, in vitro motility assays, fluorescence spectroscopy, protein expression in eukaryotic systems, and protein biochemistry and characterization. In Project #1, Dr. Berger will use a spectroscopic approach, both in solution and at the single molecule level, to determine how the lever arm tilts during the contractile cycle, and at the single molecule level, to determine how the lever arm tilts during the contractile cycle, and if the cleft opens and closes to mediate its affinity for actin. Dr. Warsaw (Project #2) will use the laser trap coupled with total internal reflectance microscopy to simultaneously measure the mechanics of single myosin molecules and either the nucleotide at its active site (single molecule fluorescence), or the orientation of the lever arm (fluorescence polarization). Project #3 (Dr. Trybus) will use a mutational approach coupled to biochemical and structural techniques (cryoelectron microscopy, crystallography) to decipher how the two heads of myosin cooperate in force and motion production, how the actomyosin interface changes during the powerstroke, and if ATP hydrolysis is required for priming of the lever arm. Dr. Lowey (Project #4) proposes to test the generality of domain movements at the actomyosin interface and in the lever arm, using cryoelectron microscopic techniques. It will also be determined if the orientation of actin and myosin affect the working stroke at the single molecule level. The long-term goal is to understand how each of the structural domains of myosin contributes to chemomechanical coupling, so that it becomes possible to derive a set of design principles for molecular motors.