The molecular basis of muscle contraction is thought to be a configurational change in the myosin head while attached to actin. Such a change could either be a conformational change within the head itself, or it may be an alteration in the binding angle made with actin. The energy for force production Is known to come from ATP hydrolysis, but while the """"""""tilting cross-bridge"""""""" hypothesis remains attractive, It is still unproven. Moreover, some recent data has suggested that the configuration of attached myosin heads may be similar in both the ATP and ADP states. This has added weight to suggestions that alternative force generation mechanisms should be considered. The main problem in testing the tilting bridge hypothesis has been the difficulty in establishing the structures of so-called weakly bound acto- myosin states that occur in the presence of ATP. A considerable amount is known about the structure of the strongly bound state that occurs in ADP or no nucleotide. However, weakly bound ATP states have been elusive because they dissociate at the low protein concentrations required for moderate resolution (approximately 3 nm) microscopy of the acto-myosin head (S1) complex. We recently found conditions that stabilize the weakly bound states and have been studying their structures by electron cryo- microscopy hydrated specimens. The micrographs already obtained show a variety of attached S1 configurations during steady- state ATP hydrolysis; such data are therefore compatible with the tilting bridge hypothesis but do not prove it. We now wish to extend this work to the study the structures of kinetically defined weak states in the ATPase cycle. If different kinetic states can be shown to have substantially different structures it will provide strong support for the tilting crossbridge hypothesis. This work will be facilitated by new methodology allowing time-resolved freezing of the electron microscope specimens to be carried out on the millisecond scale. The primary objective of this work is to obtain micrographs of biochemically defined weakly attached actomyosin states that are suitable for image processing methods. The long range goal is to obtain low resolution structures of weakly attached crossbridge states that can be used with the high resolutions crystal structures of actin and myosin to complement the actomyosin rigor structures that have been recently obtained. We believe this approach offers excellent prospects to resolve what has been arguably the single most important problem in understanding muscle function for more than two decades.
