Regulation of muscle contraction is difficult to study because it involves changes in several proteins that are in a highly organized system. Biochemical studies provide detailed information of the components but at the loss of the organization. Fiber studies preserve the structure but give little information about the function of the components. By exchanging native regulatory proteins in a single fiber with proteins containing a probe, it is possible to study the function of single components within a fiber. Fluorescent probes on troponin and tropomyosin have been shown, in solution studies, to be sensitive to calcium binding or to the fraction of actin in the active configuration. When placed into single fibers, the fluorescent probes can be used to monitor the state of the regulatory apparatus during various mechanical perturbations. This allows one to follow individual regulatory proteins in the organized structure of the fiber and to answer many questions related to regulation. The method of exchange that is used is an improvement over older methods and leaves the mechanical properties of the fibers unchanged. The plan is to study the exchange process so that it may be optimized and extended it to other proteins in striated muscle. A number of fluorescent probes will be placed on different regions of the troponin complex and the fluorescence changes that occur in fibers will be correlated with those occurring in solution under controlled conditions. In addition to the introduction of fluorescent probes, the exchange process will be used to introduce other well characterized modified or mutant troponins into fibers to study the function of particular regions of troponin. The intent is to begin by examining the effect of the mutations of the regulatory apparatus found in patients with hypertrophic cardiomyopathy. This work is part of a collaborative effort with Dr. Bernhard Brenner (mechanical studies), Dr. Leepo C. Yu (structural studies) and Dr. Yi-der Chen (mathematical modeling).
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