Mobility of the N-Terminal Segment of Rabbit Skeletal Muscle F-Actin Detected by ¹H and ¹⁹F Nuclear Magnetic Resonance Spectroscopy

Abstract

After polymerization filamentous actin (F-actin) still shows a number of rather narrow 1H 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 1H NMR spectroscopy of a highly concentrated F-actin sample. In contrast to the effects detected earlier by 1H NMR spectroscopy, 19F 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 19F 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 T2 decreases from 16.7 ms to approximately 0.6 ms. These data suggest that the highly mobile residues observed by 1H NMR spectroscopy 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 disappearing of the 1H 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 1H 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 state 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 KMI for the equilibrium between the I- and M-states is approximately 1.3 under these conditions.

Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75

“…The complementary interfaces in these proteins mapped to regions rich in basic amino acid residues [3]. These data suggest that the N-terminus of actin, which protrudes from F-actin as a highly mobile unit [34], acts as a ' fishing-rod ' in attracting positively charged surfaces of actinbinding proteins [3].…”

Cosolvent and Crowding Effects on the Temperature and Pressure Dependent Conformational Dynamics and Stability of Globular Actin

“…Line E at 2.035 ppm at ambient pressure has been assigned earlier as the methyl group of the N-terminal N-acetyl modification of actin. 48 Its chemical shift change with pressure can only be fitted with a different set of parameters (ΔG and ΔV of 5.6 kJ mol −1 and −85 mL mol −1 ), indicating that it is involved in a different transition than lines B, C, and D (Figure 7). Line E is part of the Nterminal domain from amino acid 1 to 22 that is also visible and still highly mobile after polymerization of actin to F-actin in the so-called M-(mobile) state in equilibrium with the I-(immobile) state.…”

Synthesis and characterization of Cu(II) paddlewheel complexes possessing fluorinated carboxylate ligands

“…Previous reports of 19 F NMR being used to probe metal binding in proteins describe the use of either a fluorinated substrate [6,7], fluorinated amino acids that are in close proximity to the metal [8,9], or cysteine ligands that have been chemically modified with fluorinated substituents [10]. However, to the best of our knowledge there are no reports of the use of a fluorinated amino acid that directly ligates metal ions.…”