OSMOTIC-PRESSURE PROBE OF ACTIN-MYOSIN HYDRATION CHANGES DURING ATP HYDROLYSIS

Osmotic stress in the 0.5-5 x 10(6) dyne/cm(2) range was used to pertu rb the hydration of actin . myosin . ATP intermediates during steady-s tate 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 K-M or the V-max for MgATP hydrolysi s 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 intensit ies of S1 tryptophans or of fluorescein attached to Cys-707. The acces sibility of the fluorescent ATP analog, epsilon ADP, to acrylamide que nching was also unchanged. These data suggest that none of the steps i n the ATP hydrolysis cycle involve substantial hydration changes, whic h might occur for the opening or closing of the ATP site or of other c revices in the S1 structure. In contrast, K-M for the interaction of S 1 . MgADP . P-i with actin decreased tenfold in this range of osmotic pressure, suggesting that formation of actin . S1 . MgADP . P-i involv es net dehydration of the proteins. The dehydration volume increases a s 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 . P-i was used to estimate the surface area of the bindin g interface. This estimate was consistent with the area determined fro m the atomic structures of actin and myosin, indicating that osmotic s tress is a reliable probe of actin . myosin . ATP interactions. The ap proach 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.