Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by H-1 and F-19 nuclear magnetic resonance spectroscopy

After polymerization filamentous actin (F-actin) still shows a number of rather narrow H-1 NMR signals in its Mg2+ form which are quenched when Mg2+ is replaced by Ca2+. These resonances originate from mobile residues in F-actin. For assignment of these resonances three different strategies were used, the fluorine labeling of Cys-374 by 4-(perfluoro-tert-butyl)phenyliodoacetamide, binding studies with antibodies (Fab) against the seven N-terminal amino acids of actin, and two-dimensional H-1 NMR spectroscopy of a highly concentrated F-actin sample. In contrast to the effects detected earlier by H-1 NMR spectroscopy, F-19 NMR spectroscopy of actin labeled at its C-terminal cysteine shows no significant spectral changes in dependence on the divalent ion present, In its G- (globular) form a strong, narrow F-19 resonance can be observed at 15.06 ppm (relative to the external standard trifluoroacetic acid) which is broadened substantially after polymerization of actin. At 283 K the corresponding transverse relaxation time T-2 decreases from 16.7 ms to approximately 0.6 ms, These data suggest that the highly mobile residues observed by H-1 NMR spectposcopy do not originate from the C-terminus. Binding of Fab directed against the N-terminal amino acids of actin to Mg-F-actin leads to the dissappearing of the H-1 NMR resonances assigned to a mobile domain in F-actin, This indicates that the mobile region probably comprises the N-terminal amino acids, By homonuclear two-dimensional H-1 NMR spectroscopy it was finally possible to sequentially assign the resonances of the mobile domain of F-actin. It turned out that amino acids 1-22 are in a highly mobile stare in Mg-F-actin. The nuclear Overhauser effect data indicate that, rather surprisingly, in this high mobility state some of the beta-pleated structure is still conserved. The population of F-actin protomers in the M- (mobile) state can be obtained from the NMR spectra and was determined under different experimental conditions, In the presence of 150 mM KCl approximately half of the protomers in Mg-F-actin are in the M-state, This number is largely independent of the pH in the range studied (pH 7.2-7.8) and of the temperature in range studied (283-310 K). The equilibrium constant K-M for the equilibrium between the I- and M-states is approximately 1.3 under these conditions.