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 conformational 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 measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin's enzymology. The distance between these sites is 66.8 +/- 2.3 A when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (-1.6 +/- 0.3 A) and more significantly with ADP-AlF4 (-4.6 +/- 0.2 A). During steady-state hydrolysis of ATP, the distance is temperature-dependent, 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 approximately S1-ADP > S1-ADP-BeF3 > S1-ADP-AlF4 approximately S1-ADP-Pi and that S1-ATP > S1-ADP-Pi. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulated by acto-S1-ADP.
Xiao
M
M
Physics Department and Biophysics Center, University of Illinois, Urbana, IL 61801, USA.
Li
H
H
Snyder
G E
GE
Cooke
R
R
Yount
R G
RG
Selvin
P R
PR
D000199:Actins / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004735:Energy Transfer; D005456:Fluorescent Dyes; D005895:Gizzard, Avian; D008163:Luminescent Measurements; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011789:Quantum Theory; D011817:Rabbits; D013725:Terbium
D000199:Actins; D000227:Adenine Nucleotides; D005456:Fluorescent Dyes; D018994:Myosin Light Chains; D013725:Terbium; D000244:Adenosine Diphosphate