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.
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