Dynamics of the nucleotide pocket of myosin measured by spin-labeled nucleotides.
We have used electron paramagnetic probes attached to the ribose of ATP (SL-ATP) to monitor conformational changes in the nucleotide pocket of myosin. Spectra for analogs bound to myosin in the absence of actin showed a high degree of immobilization, indicating a closed nucleotide pocket. In the Actin.Myosin.SL-AMPPNP, Actin.Myosin.SL-ADP.BeF(3), and Actin.Myosin.SL-ADP.AlF(4) complexes, which mimic weakly binding states near the beginning of the power stroke, the nucleotide pocket remained closed. The spectra of the strongly bound Actin.Myosin.SL-ADP complex consisted of two components, one similar to the closed pocket and one with increased probe mobility, indicating a more open pocket, The temperature dependence of the spectra showed that the two conformations of the nucleotide pocket were in equilibrium, with the open conformation more favorable at higher temperatures. These results, which show that opening of the pocket occurs only in the strongly bound states, appear reasonable, as this would tend to keep ADP bound until the end of the power stroke. This conclusion also suggests that force is initially generated by a myosin with a closed nucleotide pocket.
Naber
Nariman
N
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, USA. nariman.naber@ucsf.edu
Purcell
Thomas J
TJ
Pate
Edward
E
Cooke
Roger
R
D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002918:Chymotrypsin / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008954:Models, Biological; D008968:Molecular Conformation; D009218:Myosins / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry*; D011817:Rabbits; D013113:Spin Labels; D013696:Temperature; D013816:Thermodynamics
D000199:Actins; D009711:Nucleotides; D013113:Spin Labels; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D009218:Myosins