Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: The effect of nucleotides and actin

Myosin is thought to generate movement of actin filaments via a conformatio nal change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 ( S1) by using luminescence resonance energy transfer and a photoaffinity ATP -lanthanide analog. The technique allows relatively long distances to be me asured, and the label enables site-specific attachment at the active-site w ith only modest affect on myosin's enzymology. The distance between these s ites is 66.8 +/- 2.3 Angstrom when the nucleotide is ADP and is unchanged o n binding to actin. The distance decreases slightly with ADP-BeF3, (-1.6 +/ - 0.3 Angstrom) and more significantly with ADP-AlF4 (-4.6 +/- 0.2 Angstrom ). During steady-state hydrolysis of ATP, the distance is temperature-depen dent, becoming shorter as temperature increases and the complex with ADP . Pi is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP approximate to S1-AD P > S1-ADP-BeF3 > S1-ADP-AlF4 approximate to S1-ADP-P-i and that S1-ATP > S 1-ADP-Pi. The changes in distance are consistent with a substantial rotatio n of the light-chain binding domain of skeletal S1 between the prepowerstro ke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulat ed by acto-S1-ADP.