CROSS-BRIDGE MECHANICS

我们将测量通透性肌肉纤维的力学性能 与肌球蛋白结合的ATP类似物和其他配体的存在 核苷酸位置。肌肉力学性质的变化 在这些条件下的纤维将与溶液生物化学相关。 肌动球蛋白系统。机械数据还将与 用EPR探针获得的纤维在高温下活化的结构数据 同样的条件。这三组数据将一起用于 定义跨桥功能的模型，以便更好地理解 在主动收缩肌肉的过程中产生的力和运动。这个 活性纤维的力学将通过三个测量来表征。 将针对不同的伸长率测量纤维硬度以评估 进入束缚状态的附着率。光解释放笼子 磷酸盐和张力在快速缩短后的增加将 两者都探索了跨桥从低力、弱附着的过渡 状态进入高强度、强连接的PowerStroke状态。结构性 有关这些纤维的数据将从放置在以下位置的顺磁探针获得 三个地点。轻链上的探测器将监视 肌球蛋白头部的颈部区域，而针对反应性半胱氨酸的探针将 测量催化域的密度。此外，我们还将开发一部小说 一组专门放置在核苷酸位置的探针，通过使用一些 上述光亲和类似物中的一种，以确定在 核苷酸口袋的构象，以及这些变化是如何关联的 与纤维的机械状态有关。在一个相关项目中，我们将聘用 一种研究纤维力学的温度跳跃技术 在高温(15-39摄氏度)下激活。最近的结果表明， 在20摄氏度以上的温度下，几个力学参数表现为 与低温下不同。尤其是pH值的下降， 人们认为在疲劳的肌肉中发挥主要作用的人，已经大大减少了 在更高的温度下会产生影响。综合在一起，在 上述实验将有助于定义一些 在跨桥循环中的状态，使我们能够更现实地 这种相互作用的模型，使我们更好地理解 肌肉活动的复杂现象和对肌肉的更深层次的理解 疲惫。

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.