Rotation of Single Cross-Bridge in Skeletal Muscle Fiber

描述（由申请人提供）：目前骨骼肌收缩的假设是，“燃料”分子（ATP）与肌球蛋白活性位点的结合诱导催化结构域（分子的酶活性部分）的局部构象变化。这种变化被机械放大，并导致调节结构域的主要旋转-肌球蛋白末端的一个长部分，是酶惰性的。调节域的旋转导致力和运动的产生。这种旋转与肌球蛋白活性部位发生的化学反应有关。这项提议的目的是在收缩肌纤维的单个交叉桥中测试这一假设。共聚焦显微镜被修改，以允许从一个小的人口（约10）的跨桥测量。同时测量调节结构域的旋转和酶活性。通过测量放置在调控域内的战略位置的探针的荧光的各向异性来研究旋转。在瞬时收缩期间（通过从笼中突然释放ATP产生）或在稳态收缩期间（通过使用相关光谱法）测量各向异性。通过由近场探针发出的光激发的磷酸盐结合蛋白的荧光来测量酶活性。将各向异性和酶信号互相关以建立它们的因果关系。这项工作的意义在于，流行的假设将被测试，第一次在一个单一的工作肌跨桥。这有望为骨骼肌收缩机制提供明确的答案。

DESCRIPTION (provided by applicant): The current hypothesis of contraction of skeletal muscle is that the binding of a "fuel" molecule (ATP) to an active site of myosin induces a local conformational change in the catalytic domain --an enzymatically active part of a molecule. This change is mechanically amplified and leads to a major rotation of the regulatory domain -- a long part at the end of myosin that is enzymatically inert. The rotation of the regulatory domain results in the generation of force and movement. The rotation is coupled to the chemical events occurring at the active site of myosin. The aim of this proposal is to test this hypothesis in a single cross-bridge of contracting muscle fiber. A confocal microscope is modified to allow measurements from a small population (approximately 10) of cross-bridges. The rotation of the regulatory domain and the enzymatic activity are measured simultaneously. The rotation is studied by measuring the anisotropy of fluorescence of probes placed at strategic positions within the regulatory domain. The anisotropy is measured either during transient contraction (created by suddenly releasing ATP from a cage) or during steady-state contraction (by using correlation spectroscopy method). The enzymatic activity is measured by fluorescence of phosphate binding protein excited by light emerging from a Near-Field probe. The anisotropy and enzymatic signals are cross-correlated to establish their causal relationship. The significance of this work is that the prevailing hypothesis will be tested, for the first time in a single cross-bridge of working muscle. This is expected to provide definitive answers about the mechanism of contraction of skeletal muscle.