Osmotic pressure probe of actin-myosin hydration changes during ATP hydrolysis.
Osmotic stress in the 0.5-5 x 10(6) dyne/cm2 range was used to perturb the hydration of actin-myosin-ATP intermediates during steady-state hydrolysis. Polyethylene glycol (PEG) (1000 to 4000 Da), in the 1 to 10 wt% range, which does not cause protein precipitation, did not significantly affect the apparent KM or the Vmax for MgATP hydrolysis by myosin subfragment 1 (S1) alone, nor did it affect the value for the phosphate burst. Consistent with the kinetic data, osmotic stress did not affect nucleotide-induced changes in the fluorescence intensities of S1 tryptophans or of fluorescein attached to Cys-707. The accessibility of the fluorescent ATP analog, epsilon ADP, to acrylamide quenching was also unchanged. These data suggest that none of the steps in the ATP hydrolysis cycle involve substantial hydration changes, which might occur for the opening or closing of the ATP site or of other crevices in the S1 structure. In contrast, KM for the interaction of S1.MgADP.Pi with actin decreased tenfold in this range of osmotic pressure, suggesting that formation of actin.S1.MgADP.Pi involves net dehydration of the proteins. The dehydration volume increases as the size of the PEG is increased, as expected for a surface-excluded osmolyte. The measured dehydration volume for the formation of actin.S1.MgADP.Pi was used to estimate the surface area of the binding interface. This estimate was consistent with the area determined from the atomic structures of actin and myosin, indicating that osmotic stress is a reliable probe of actin.myosin.ATP interactions. The approach developed here should be useful for determining osmotic stress and excluded volume effects in situ, which are much larger than those of typical in vitro conditions.
Highsmith
S
S
Department of Biochemistry, School of Dentistry, University of the Pacific, San Francisco, California 94115-2399, USA. shighsmith@uop.edu
Duignan
K
K
Cooke
R
R
Cohen
J
J
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D015394:Molecular Structure; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D009997:Osmotic Pressure; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D013816:Thermodynamics; D014867:Water / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D010710:Phosphates; D014867:Water; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins