Structural Dynamics Of Actomyosin Motility

肌动球蛋白运动的结构动力学

• 批准号: 7528513
• 负责人: YALE E GOLDMAN
• 金额: $ 47.49万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7528513
• 关键词: Accounting; Actins; Active Sites; Actomyosin; Actomyosin Adenosinetriphosphatase; Address; Arsenicals; Binding; Biochemical; Biochemical Reaction; Biological Assay; Blindness; Calmodulin; Cell division; Chemicals; Chemotaxis; Compatible; Complex; Contractile Proteins; Cysteine; Cytoskeletal Filaments; Data; Dependence; Detection; Development; Development, Other; Dimensions; Disease; Dissociation; Engineering; Event; Fatigue; Feedback; Fluorescence; Fluorescence Polarization; Fluorescent Probes; Generations; Head; Hypertension; Hypertrophic Cardiomyopathy; Isometric Exercise; Kinetics; Knowledge; Label; Light; Link; Localized; Measurement; Measures; Mechanical Stress; Mechanics; Methods; Microfilaments; Microscope; Microscopy; Molecular; Molecular Motors; Monitor; Motion; Motor; Muscle; Muscle Weakness; Myosin ATPase; Myosin Type II; Nature; Neoplasm Metastasis; Neurons; Polarization Microscopy; Positioning Attribute; Process; Protein Isoforms; Proteins; Public Health; Rate; Recombinants; Reporting; Research; Resolution; Slide; Space Perception; Speed; Striated Muscles; Structural Protein; System; Techniques; Testing; Therapeutic; Time; Tissues; Ursidae Family; Use of New Techniques; Validation; Work; Working stroke; base; cell motility; congenital deafness; design; design and construction; fundamental research; improved; inorganic phosphate; monomer; myosin VI; nanometer; neoplastic; nervous system disorder; new technology; novel; novel diagnostics; optical traps; relating to nervous system; research study; single molecule; size; technique development

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.

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能的分子机制，并在分子水平上获得机械、生化和结构事件之间的精确关联。新方法将应用于分离的肌肉肌球蛋白和非常规肌球蛋白分子马达，以探讨收缩蛋白的生化反应、跨桥循环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至运动结构域和肌球蛋白头中的轻链亚基。这些成分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。旋转和平移运动的时间分辨率的提高将使分子步骤中的事件得以确定。使用新的“视差视图”技术对 x、y 和 z 方向上的分子位置进行纳米分辨率跟踪将揭示分子步进的机制和沿着细胞骨架丝的路径选择。红外光阱具有高速反馈功能，可将肌动蛋白固定到位，将与单分子 polTIRF 显微镜相结合，直接评估机械应力和应变对与化学机械转导相关的步进速率和蛋白质方向变化的影响。迈步目标选择的能量学和概率性质将根据头部角度、生化状态和步长分布的方向和力依赖性来确定。实验将在从肌肉组织分离的常规肌球蛋白和从神经组织和重组表达系统分离的非肌肉肌球蛋白上进行。该项目的结果将显着增进对细胞运动过程的了解，从而更好地了解横纹肌的正常和病理状态、神经元发育和其他类型的细胞运动。公共健康相关性：肌球蛋白分子马达与许多疾病有关、改变或受损，例如肌肉无力和疲劳、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，例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为产生新的诊断和治疗措施提供基本的了解。