CROSS-BRIDGE MECHANICS

我们将测量透化肌纤维的力学， 存在ATP类似物和其他与肌球蛋白结合的配体 核苷酸位点。肌肉力学特性的改变 纤维在这些条件下将与溶液生物化学 肌动球蛋白系统。机械数据还将与 用EPR探针获得的纤维结构数据， 同等条件这三组数据将用于 定义跨桥功能的模型，以便更好地理解 肌肉收缩时产生的力量和运动。的 活性纤维的力学将通过三个测量来表征。 将测量不同拉伸速率的纤维刚度，以评估 结合成束缚态的比率。光解释放笼状 磷酸盐和快速缩短后张力增加的速率将 两者都探讨了跨桥从低力、弱附着 状态转化为高强度、强连接的动力冲程状态。结构 这些纤维上的数据将从放置在 三个网站。轻链上的探针将监测轻链的方向。 肌球蛋白头部的颈部区域，而反应性半胱氨酸的探针将 测量催化结构域的温度。此外，我们将开发一个新的 一组探针，通过使用一些 以上光亲和性类似物，以确定发生什么变化， 核苷酸口袋的构象，以及这些变化如何相互关联 与纤维的机械状态有关。在一个相关的项目中， 研究纤维力学的温度跳跃技术 在高温（15-39摄氏度）下活化。最近的结果显示， 在温度高于20摄氏度时， 与低温下不同。特别是pH值的降低， 被认为在疲劳的肌肉中起主要作用， 在较高温度下的效果。综合上述结果， 上述实验将有助于确定一些动力学和能量 在跨桥周期的国家，使我们能够作出更现实的 这种相互作用的模型，导致更好地了解 活动肌肉的复杂现象和对肌肉的更深入了解 疲劳

We shall measure the mechanics of permeabilized muscle fibers in the presence of ATP analogs and other ligands which bind to the myosin nucleotide site. The alterations in the mechanical properties of muscle fibers under these conditions will be correlated with solution biochemistry of the actomyosin system. The mechanical data will also be combined with structural data obtained with EPR probes for fibers activated under the same conditions. Together these three sets of data will be used to define models of cross-bridge function in order to better understand the production of force and motion in actively contracting muscle. The mechanics of active fibers will be characterized by three measurements. Fiber stiffness will be measured for different rates of stretch to assess the rates of attachment into bound states. Photolytically released caged phosphate and the rate of tension increase following rapid shortening will both probe the transition of cross-bridges from low-force, weakly-attached states into high-force, strongly-attached powerstroke states. Structural data on these fibers will be obtained from paramagnetic probes placed at three sites. Probes on the light chains will monitor the orientation of the neck region of the myosin head, while probes at a reactive cysteine will measure that of the catalytic domain. In addition we will develop a novel set of probes placed specifically at the nucleotide site by employing some of the above photoaffinity analogs to determine what changes occur in the conformation of the nucleotide pocket, and how these changes are correlated with the mechanical state of the fiber. In a related project we will employ a temperature jump technique to investigate the mechanics of fibers activated in high (15-39 degree C) temperatures. Recent results show that at temperatures above 20 degree C several mechanical parameters behave differently than at low temperatures. In particular, a decrease in pH, thought to play a major role in fatigued muscle, has a greatly reduced effect at the higher temperatures. Together the results obtained in the above experiments will help define the kinetics and energetics of a number of states in the cross-bridge cycle, allowing us to make more realistic models of this interaction, leading to a better understanding of the complex phenomena of active muscle and to a deeper understanding of muscle fatigue.