9404705 Mendelson The control of vertebrate muscle contraction is regulated by the binding and unbinding of Ca2+ to the thin-filament protein troponin (Tn). We will study these changes and also those that occur when myosin heads bind to the thin filament. Neutron scattering and diffraction will be used to study filamentous samples in vitro. Selective deuteration of one or more of the members of a complex will allow either the protonated or deuterated member(s) to be rendered "invisible" to neutrons by solvent contrast matching or a related method. Structural changes of the visible components can thus be investigated in situ with little ambiguity. The average cross-helix distance between the tropomyosin (Tm) molecules will be investigated +Ca2 and both with and without myosin subfragment 1 (S1). Tm ordering in thin filaments will be investigated +Ca2. Changes in the structure of F-actin as a member of the thin filament will be investigated +Ca2+. If such changes are found, the structure of F-actin in situ will be refined to understand the nature of these changes. The average cross-helix separation between whole Tn and Tn subunits (TnC, TnI and TnT) will be studied +Ca2+ and +S1. We will search for circumferential movement of Tn +Ca2+. We will measure the in situ shape of the TnI-TnC complex and search for shape changes +Ca2+ and +S1. We will determine the orientation of TnC in thin filaments and study its changes upon Ca2+ binding. We expect that the proposed experiments will important new information about structural changes occurring in the regulation of vertebrate muscle contraction and will help to resolve current controversies. %%% The control of vertebrate muscle contraction is regulated by the binding and unbinding of Ca2+ to the thin filament protein troponin. Upon Ca2+ binding, structural changes occur in the thin filament which mediate the interaction of the proteins responsible for muscle contraction (myosin and actin) when fuel (ATP) is present. Neutron scattering will be used to study thin filament samples. Selective deuteration of one or more of the members of a complex will allow the (native) protonated member(s) to be rendered effectively "invisible" to neutrons by varying the D2O content of the sample. Structural changes of the visible components, +Ca2+, will then be investigated in situ with little ambiguity. We believe that studies such as these, in addition to increasing our knowledge of vertebrate muscle control, can help us understand other forms of biological control and, in particular, how the binding of small ions to proteins can cause large-scale macromolecular and physiological changes. Development of some of the methodologies to be employed, such as deuteration of cloned proteins and neutrons scattering techniques, may be related to national efforts in biotechnology. ***
