Our main goal is to identify the structural changes that accompany the myosin power stroke and cause force generation in muscle, to a resolution of about 5 nm in space and about 5 ms in time. To correlate the structure, mechanics and timing of crossbridge action in the highly ordered insect flight muscle (IFM) of Lethocerus, we will coordinate electron microscopy, X-ray diffraction and physiological measurements. Rapid structural transitions will be time-resolved by quick freezing, and examined by thin-section EM and image reconstruction of freeze- substituted muscle, supported by time-slicing synchrotron X-ray diffraction. Concerted crossbridge transitions will be triggered by photolysis of """"""""caged"""""""" (inert photolabile precursors of) substrate or signal molecules, and also by sudden length steps that produce stretch-activation in IFM. The steady-state equilibrium structures of quick-frozen IFM fibers in rigor, relaxed and isometrically contracting states will be established first, to be followed by studies of rapid transitions. We will time-resolve structural changes of the crossbridge cycle by EM of fibers quick-frozen at 15, 30, 50, 100 and 300 ms after photochemical triggering of the rigor-to-relaxed and rigor-to-active transitions. Stretch-activation of the relaxed-to-active transition will be similarly time-resolved. Rigor contractions will be a special focus, if we can achieve rapid single-turnover relaxed-to-rigor transitions. X-ray diffraction including time-slicing studies at synchrotron X-ray sources will characterize the same photolytically or stretch-activated transitions. We will try to capture quicker and better synchronized transitions by quick-freezing contracting fibers during force transients that follow sudden length steps. Quick release may synchronize a quick-recovery power stroke. Quick stretch should drag attached bridges """"""""backwards"""""""" into their highest force-producing state. Structure of """"""""pre-force"""""""" attached crossbridge state(s) will be explored by EM and X-ray comparison of fibers that develop stiffness but little or no force/shortening in, high Ca2+ after equilibration with glycol-AMPPNP, ATPgammaS, ADPAIF3, PrNANTP and possibly ADP-Vi. We will collaborate in seeking spin-label strategies and probes that may report closer agreement with EMs and X-ray about orientations of the bulk of averaged myosin head mass, apparently uniform (at 90 degrees) in relaxed, and multiple (at 48 degrees, 77 degrees and 90 degrees) in rigor. Collaborative mutational analysis of actin-crossbridge interaction in site-directed actin-mutant Drosophila flight muscle will include our ultrastructural analysis and participation in in vitro motility studies, and 3D reconstructions of normal and reverse rigor chevrons when possible.
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