The primary goal of this project is to provide the applicant with rigorous training in muscle biochemistry and biophysics, to prepare him for a career as an independent investigator in this field. His strong background in cellular physiology will be augmented by training in muscle biophysics, with particular emphasis on electron paramagnetic resonance of contracting muscle, which is a particular strength of the sponsor's laboratory. This training program focuses on a research project that is central to the sponsor's NIH-funded research. The overall goal of this project is to answer fundamental questions about the role of protein phosphorylation and dynamics in the function of smooth muscle, with particular emphasis on understanding the """"""""latch"""""""" state of smooth muscle, which has great potential biomedical importance.
AIMS : (1) Develop a spectroscopic method, involving spin-labels and electron paramagnetic resonance (EPR), to measure accurately, in real time, the extent of phosphorylation of the regulatory light chain (RLC) in skinned smooth muscle fibers. This method is based closely on previous publications from the sponsor's laboratory. (2) Use this EPR method to determine quantitatively the relationship between phosphorylation level, muscle force, and the distribution of myosin molecules between weak and strong actin binding conformational states, in isometrically contracting smooth muscle. Particular emphasis will be placed on conditions under which the muscle enters the latch state, and to determine the effect of RLC phosphorylation on this state. (3) The third aim probes the effect of inorganic phosphate in perturbing the mechanochemical kinetics of the contraction process. By completing these aims, we will test directly the key hypotheses for the molecular mechanism of the latch state in smooth muscle.
We are testing fundamental questions about the function and malfunction of smooth muscle regulation. The answers to these questions will provide crucial insights into the underlying biochemistry and biophysics responsible for abnormal smooth muscle contraction in asthma and hypertension. Our molecular measurements in contracting muscle can provide the basis for evaluation of drugs and other therapeutic approaches. ? ? ?
|Muretta, Joseph M; Behnke-Parks, William M; Major, Jennifer et al. (2013) Loop L5 assumes three distinct orientations during the ATPase cycle of the mitotic kinesin Eg5: a transient and time-resolved fluorescence study. J Biol Chem 288:34839-49|
|Muretta, Joseph M; Petersen, Karl J; Thomas, David D (2013) Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain. Proc Natl Acad Sci U S A 110:7211-6|
|Wilson, Daniel J; Shi, Ce; Duckworth, Benjamin P et al. (2011) A continuous fluorescence displacement assay for BioA: an enzyme involved in biotin biosynthesis. Anal Biochem 416:27-38|
|Muretta, Joseph M; Kyrychenko, Alexander; Ladokhin, Alexey S et al. (2010) High-performance time-resolved fluorescence by direct waveform recording. Rev Sci Instrum 81:103101|