Conformational selection during weak binding at the actin and myosin interface.
The molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5 nm) were detected that are consistent with internal conformational changes of the myosin molecule, but not with extreme differences in the average lever arm position suggested by some atomic models. These results were confirmed by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin. To examine the participation of actin in the powerstroke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the presence of nucleotide transition state analogs. The efficiency of energy transfer was much greater in the presence of ADP-AlF(4), ADP-BeF(x), and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached cross-bridges that appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin.
Xu
J
J
Department of Biological Sciences, University of North Texas, Denton, Texas 76203-5220 USA.
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D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003196:Computer Graphics; D004228:Dithionitrobenzoic Acid; D007140:Immunoglobulin Fab Fragments; D007700:Kinetics; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013816:Thermodynamics
D000199:Actins; D007140:Immunoglobulin Fab Fragments; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D009218:Myosins