Phosphorus-31 nuclear magnetic resonance studies of the adenosine 5'-triphosphate-calcium-G-actin complex.
Phosphorus-31 nuclear magnetic resonance spectra of the adenosine 5'-triphosphate-calcium-G-actin complex were obtained, and the resonances of the three phosphates of the protein-bound ATP were detected. The exchange of the ATP between its protein-bound and free states were found to be slow on the NMR time scale, with an exchange rate of less than 480 s-1 at pH* 7.8, 4 degrees C. The line width of the protein-bound gamma-phosphate resonance (corrected for spin-spin splitting by the beta phosphate) was used to calculate a rotational correlation time for the G-actin-bound ATP. With the assumption that chemical shift anisotropy is the dominant relaxation mechanism at 109.29 MHz and that the chemical shielding tensor for pyrophosphate serves as a good model for the gamma phosphate of the bound ATP, a correlation time of 60 ns was estimated. Since the theoretical correlation time of a globular protein the size of G-actin is 36 ns, the line width of the bound gamma-phosphate resonance is consistent with that expected for ATP bound to G-actin without large-scale rapid internal mobility. The addition of 1.5 M urea to the ATP-Ca-G-actin complex caused exchange broadening of the gamma and beta phosphates, but no effect on the alpha phosphate. This indicates an increase in the rate of exchange for the beta and gamma phosphates between the protein-buried and solvent-exposed environments at 1.5 M urea. At 6 M urea, the intensities of the protein-bound ATP resonances were greatly reduced, and the intensities of the free ATP resonances were greatly increased, indicative of complete protein unfolding and liberation of protein-bound ATP.
D000199:Actins; D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D002118:Calcium; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D000255:Adenosine Triphosphate; D002118:Calcium