MYOSIN DYNAMICS DURING THE CONTRACTILE CYCLE

这个项目的主题是了解分子运动是如何工作的，特别是阐明在收缩周期中肌球蛋白的结构转变。为了解决这些问题，阐明在收缩周期中肌球蛋白发生的结构转变。为了解决这些问题，这四个项目汇集了一系列最先进的技术，包括单分子力学测量、体外运动测定、荧光光谱、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博士将使用光谱学方法，在溶液和单分子水平上，确定杠杆臂在收缩周期中是如何倾斜的，在单分子水平上，确定杠杆臂在收缩周期中是如何倾斜的，以及裂缝是否打开和关闭以调节其对肌动蛋白的亲和力。华沙博士（项目#2）将使用激光陷阱与全内反射显微镜相结合，同时测量单个肌球蛋白分子的力学，以及其活性位点的核苷酸（单分子荧光）或杠杆臂的方向（荧光偏振）。项目#3 （Trybus博士）将使用一种突变方法，结合生化和结构技术（低温电子显微镜、晶体学）来破译肌凝蛋白的两个头部如何在力量和运动产生中合作，肌动球蛋白界面在动力冲力过程中如何变化，以及杠杆臂的启动是否需要ATP水解。Lowey博士（项目#4）建议使用低温电子显微镜技术测试肌动球蛋白界面和杠杆臂区域运动的普遍性。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卒中。长期目标是了解肌球蛋白的每个结构域如何促进化学-力学耦合，从而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.