Structural dynamics of the myosin relay helix by time-resolved EPR and FRET.
We have used two complementary time-resolved spectroscopic techniques, dipolar electron-electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF(x)) or ADP.P(i) (ADP.AlF(4)) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF(4) was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition.
Agafonov
Roman V
RV
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Negrashov
Igor V
IV
Tkachev
Yaroslav V
YV
Blakely
Sarah E
SE
Titus
Margaret A
MA
Thomas
David D
DD
Nesmelov
Yuri E
YE
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D013113:Spin Labels
D013113:Spin Labels; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins