Structural Dynamics Of Actomyosin Motility

描述（由申请人提供）：本研究的总体目标是了解肌动球蛋白运动系统将化学能转化为机械功的分子机制，并在分子水平上获得机械、生物化学和结构事件之间的精确相关性。新的方法将被应用到分离的肌肉肌球蛋白和非常规肌球蛋白分子马达，以探测收缩蛋白的生化反应，跨桥周期的基本机械步骤和相应的结构运动之间的关系。双功能或双砷荧光探针分子将以已知的方向稳定地结合到肌球蛋白头中的马达结构域和轻链亚基。这些组件的空间方向和平移位置将通过新型单分子荧光偏振全内反射（polTIRF）显微镜以高时间分辨率进行监测，以确定特定蛋白质结构变化的动态。提高旋转和平移运动的时间分辨率将使分子步骤期间的事件能够被确定。使用新的“视差视图”技术在x、y和z方向上对分子位置的纳米分辨率跟踪将揭示分子步进和沿沿着细胞骨架丝的路径选择的机制。一个红外光学陷阱，与高速反馈的肌动蛋白钳位，将与单分子polTIRF显微镜直接评估机械应力和应变的步进率和蛋白质取向的变化，涉及化学机械转导的影响。步进目标选择的能量学和概率性质将从头部角度、生化状态和步长分布的方向和力依赖性来确定。将对从肌肉组织分离的常规肌球蛋白和从神经组织分离的非肌肉肌球蛋白以及重组表达系统进行实验。从这个项目的结果应显着推进细胞运动过程的知识，从而带来了横纹肌，神经元发育和其他类型的细胞运动的正常和病理状态的更好的理解。公共卫生相关性：肌球蛋白分子马达参与、改变或损害许多疾病，如肌无力和疲劳、肥厚性心肌病、高血压、细胞分裂、趋化性、肿瘤侵袭和转移，以及许多神经系统疾病，如特定形式的发育缺陷和先天性耳聋和失明。因此，对这些蛋白质的基础研究将提供基本的了解，从而能够产生新的诊断和治疗措施。

DESCRIPTION (provided by applicant): The overall aims of this research are to understand the molecular mechanism by which actomyosin motility systems convert chemical energy into mechanical work, and to obtain a precise correlation between the mechanical, biochemical and structural events at the molecular level. Novel methods will be applied to isolated muscle myosin and unconventional myosin molecular motors to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. Bifunctional or bi-arsenical fluorescent probe molecules will be stably bound with known orientation to the motor domains and to light chain subunits in the myosin heads. The spatial orientation and translational position of these components will be monitored at high time resolution by novel single-molecule fluorescence polarization, total internal reflection (polTIRF) microscopy to determine the dynamics of specific protein structural changes. Increased time resolution of the rotational and translational motions will enable events during molecular steps to be determined. Nanometer resolution tracking of the molecular position in the x, y and z directions, using a new "Parallax View" technique will disclose mechanisms of molecular stepping and choice of path along the cytoskeletal filament. An infrared optical trap, with high-speed feedback to clamp the actin in place, will be combined with single-molecule polTIRF microscopy to directly evaluate the influence of mechanical stress and strain on stepping rates and protein orientation changes that relate to chemo-mechanical transduction. The energetics and probabilistic nature of stepping target selection will be determined from the orientation and force dependence of the distributions of head angle, biochemical state and step size. The experiments will be carried out on conventional myosin isolated from muscle tissue and on non-muscle myosins isolated from neural tissue and recombinant expression systems. Results from this project should significantly advance knowledge of cell motility processes and thus bring a greater understanding of both normal and pathological states of striated muscle, neuronal development and other types of cell motility. PUBLIC HEALTH RELEVANCE: Myosin molecular motors are involved, altered or compromised in many diseases, such as muscle weakness and fatigue, hypertrophic cardiomyopathy, hypertension, cell division, chemotaxis, neoplastic invasiveness and metastasis, and in many neurological diseases, such as specific forms of developmental deficits and congenital deafness and blindness. Fundamental research on these proteins thus will provide the basic understanding to enable generation of new diagnostic and therapeutic measures.