Phosphorylation regulates the ADP-induced rotation of the light chain domain of smooth muscle myosin.
We have observed the effects of MgADP and thiophosphorylation on the conformational state of the light chain domain of myosin in skinned smooth muscle. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the orientation of spin probes attached to the myosin regulatory light chain (RLC). Two spectral states were seen, termed here "intermediate" and "final", that are distinguished by a approximately 24 degrees axial rotation of spin probes attached to the RLC. The two observed conformations are similar to those found previously for smooth muscle myosin S1; the final state corresponds to the major conformation of S1 in the absence of ADP, while the intermediate state corresponds to the conformation of S1 with ADP bound. Light chain domain orientation was observed as a function of the MgADP concentration and the extent of RLC thiophosphorylation. In rigor (no MgADP), LC domains were distributed equally between the intermediate state and the final state; upon addition of saturating (3.5 mM) MgADP, about one-third of the LC domains in the final state rotated approximately 20 degrees axially to the intermediate state. The progression of the change in populations was fit to a simple binding equation, yielding an apparent dissociation constant of approximately 110 microM for skinned smooth muscle fibers and approximately 730 microM for thiophosphorylated, skinned smooth muscle fibers. These observations suggest a model that explains the behavior of "latch bridges" in smooth muscle.
Gollub
J
J
Graduate Group in Biophysics, Department of Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco 94143-0448, USA.
Cremo
C R
CR
Cooke
R
R
D008841:Actin Cytoskeleton / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002645:Chickens; D003497:Cyclic N-Oxides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D005895:Gizzard, Avian; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism; D009130:Muscle, Smooth / Q000737:chemistry / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013997:Time Factors
D003497:Cyclic N-Oxides; D020409:Molecular Motor Proteins; D018994:Myosin Light Chains; D010446:Peptide Fragments; D013113:Spin Labels; C003028:N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; D000244:Adenosine Diphosphate