Rotation of Single Cross-Bridge in Skeletal Muscle Fiber

描述(申请人提供)：目前关于骨骼肌收缩的假说是，“燃料”分子(ATP)与肌球蛋白活性部位的结合会导致催化区域的局部构象变化--催化区域是分子中具有酶活性的部分。这种变化是机械放大的，并导致调节域的主要旋转--肌球蛋白末端的长部分在酶的作用下是惰性的。调控结构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事件相耦合。这项提议的目的是在收缩肌肉纤维的单一桥梁上检验这一假设。对共聚焦显微镜进行了改进，使之可以从一小群(大约10个)的交叉桥中进行测量。同时测量了调节域的旋转和酶的活性。通过测量放置在调节域内的战略位置的探针的荧光各向异性来研究旋转。各向异性是在瞬时收缩期间(通过突然从笼子中释放ATP而产生)或在稳态收缩期间(使用相关光谱学方法)测量的。酶活性是通过近场探头发出的光激发的磷酸结合蛋白的荧光来测量的。各向异性和酶信号相互关联以建立它们的因果关系。这项工作的意义在于，盛行的假说将首次在工作肌肉的单一桥梁上得到检验。这有望为骨骼肌收缩的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.