Muscle cross-bridge mechanism investigated by fluorescence lifetime imaging microscopy of myosin essential light chain.

了解肌肉收缩过程中化学能如何转化为机械运动的关键是了解肌球蛋白马达中与动力冲程相关的构象事件。这个跨学科的研究项目的目的是连接ATP水解的生化序列和肌球蛋白的构象变化，导致其位移沿着肌动蛋白丝在肌纤维。我们将使用荧光寿命成像显微镜（FLIM）的新技术来研究肌球蛋白酶动力学及其与有组织生物系统中结构状态的关系-透化肌纤维，荧光探针连接到肌球蛋白的特定位置，特别是肌球蛋白必需轻链（ELC）。荧光寿命提供了关于细胞中荧光团的环境的信息，其优点在于，与荧光强度的成像相比，不依赖于荧光团浓度。该项目将率先在生物分子研究中使用时间分辨FLIM，并且使用横纹肌细胞提供的空间平均机会应该允许无与伦比的空间和时间分辨率。这项工作很重要，因为它将使人们能够更好地理解肌肉中能量转换的基本方面，并将展示外部施加的力与酶催化位点的行为之间的相互作用。我们将利用帝国理工物理学院的FLIM专业知识和NIMR的荧光光谱学。在分离的肌纤维中使用基于香豆素的荧光团标记的ELC的初步结果表明，基因表达的ELC可以以高效率和特异性引入肌纤维中，同时保持肌肉功能。我们还发现，香豆素-ELC在僵直肌肉中的荧光寿命显著低于放松肌肉，从而为进一步研究提供了一个令人兴奋的平台。项目的主要目标是：1.检测在渗透性纤维的收缩和松弛期间附着于ELC的荧光团的荧光寿命; 2.确定ELC构象的ATP和应变依赖性变化，并将这些分子事件与力和ATP水解的改变相关联; 3.研究ELC不同EF-手基序上探针的荧光性质，以获得有关构象变化的几何信息。

The key to understanding how chemical energy is converted into mechanical movement during muscle contraction is to follow conformational events associated with the power stroke in the myosin motor. The aim of this interdisciplinary research project is to link the biochemical sequence of ATP hydrolysis and conformational changes in myosin leading to its displacement along actin filaments in muscle fibres. We will use a novel technology of Fluorescence Lifetime Imaging Microscopy (FLIM) to investigate myosin enzyme kinetics and its relation to structural states in organised biological systems - permeabilised muscle fibres, with fluorescent probes attached to specific locations in myosin, in particular in the myosin essential light chain (ELC). Fluorescence lifetime provides information about the environment of a fluorophore in a cell, with the advantage, over imaging of fluorescence intensity, of independence from fluorophore concentration. The project will pioneer the use of time-resolved FLIM in biomolecular research and the spatial averaging opportunities provided by the use of striated muscle cells should allow unparalleled spatial and time resolution. The work is important because it will enable better understanding of the fundamental aspects of energy conversion in muscle, and will demonstrate the interaction between externally applied forces and the behaviour of an enzyme's catalytic site. We shall take advantage of FLIM expertise from Physics at Imperial and fluorescence spectroscopy from NIMR. Preliminary results using ELC labelled with coumarin-based fluorophore in isolated muscle fibres showed that genetically expressed ELC can be introduced in muscle fibres with high effectiveness and specificity, while maintaining muscle function. We also showed that the fluorescence lifetime of coumarin-ELC in rigor muscle is significantly lower than in relaxed muscle, thus providing an exciting platform for further investigation. The project major goals are: 1. to detect the fluorescence lifetime of fluorophores attached to ELC during contraction and relaxation of permeabilised fibres; 2. to determine the ATP and strain dependent changes in ELC conformation and to correlate these molecular events with alterations in force and in ATP hydrolysis; 3. to study the fluorescent properties of probes attached to different EF-hand motifs of ELC to gain detailed information about the geometry of the conformational changes.