G-Actin is a globular protein (Mr 42 300) known to have three cysteine residues that are at least partially exposed and chemically reactive (Cys-10, -284, and -374). When G-actin was reacted with 3-bromo-1,1,1-trifluoropropanone, three resolvable 19F resonances were observed in the 19F NMR spectrum. This fluorinated G-actin derivative remained fully polymerizable, and its 31P NMR spectrum was not significantly different from that of unmodified G-actin, indicating that the chemical modification did not denature the actin and the modified residues do not interfere with the extent of polymerization or the binding of adenosine 5'-triphosphate. One of the three 19F resonances was assigned to fluorinated Cys-374 on the basis of its selective reaction with N-ethylmaleimide. This resonance was dramatically broadened after polymerization of fluorinated G-actin, while the other two resonances were not markedly broadened or shifted. Thus, Cys-10 and -284 are not involved in or appreciably affected by the polymerization of G-actin, while the mobility of the 19F label at Cys-374 is markedly reduced.
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.
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