Mobility of the N-Terminal Segment of Rabbit Skeletal Muscle F-Actin Detected by 1H and 19F Nuclear Magnetic Resonance Spectroscopy
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- 作者：Daniela Heintz ; Harry Kany ; and Hans Robert Kalbitzer
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：October 1, 1996
- 年：1996
- 卷：35
- 期：39
- 页码：12686 - 12693
- 全文大小：493K
- 年卷期：v.35,no.39(October 1, 1996)
- ISSN：1520-4995
文摘
After polymerization filamentous actin (F-actin) still shows anumber of rather narrow 1HNMR signals in its Mg2+ form which are quenched whenMg2+ is replaced by Ca2+. Theseresonancesoriginate from mobile residues in F-actin. For assignment of theseresonances three different strategieswere used, the fluorine labeling of Cys-374 by4-(perfluoro-tert-butyl)phenyliodoacetamide, bindingstudieswith antibodies (Fab) against the seven N-terminal amino acids ofactin, and two-dimensional 1H NMRspectroscopy of a highly concentrated F-actin sample. In contrastto the effects detected earlier by 1HNMR spectroscopy, 19F NMR spectroscopy of actin labeled atits C-terminal cysteine shows no significantspectral changes in dependence on the divalent ion present. In itsG- (globular) form a strong, narrow 19Fresonance can be observed at 15.06 ppm (relative to the externalstandard trifluoroacetic acid) which isbroadened substantially after polymerization of actin. At 283 Kthe corresponding transverse relaxationtime T2 decreases from 16.7 ms to approximately0.6 ms. These data suggest that the highly mobileresidues observed by 1H NMR spectroscopy do not originatefrom the C-terminus. Binding of Fab directedagainst the N-terminal amino acids of actin to Mg-F-actin leads tothe dissappearing of the 1H NMRresonances assigned to a mobile domain in F-actin. This indicatesthat the mobile region probably comprisesthe N-terminal amino acids. By homonuclear two-dimensional1H NMR spectroscopy it was finally possibleto sequentially assign the resonances of the mobile domain of F-actin.It turned out that amino acids1-22 are in a highly mobile state in Mg-F-actin. The nuclearOverhauser effect data indicate that,rather surprisingly, in this high mobility state some of the
middle">-pleated structure is still conserved. Thepopulation of F-actin protomers in the M- (mobile) state can beobtained from the NMR spectra and wasdetermined under different experimental conditions. In thepresence of 150 mM KCl approximately halfof the protomers in Mg-F-actin are in the M-state. This numberis largely independent of the pH in therange studied (pH 7.2-7.8) and of the temperature in range studied(283-310 K). The equilibrium constantKMI for the equilibrium between the I- andM-states is approximately 1.3 under these conditions.
middle">-pleated structure is still conserved. Thepopulation of F-actin protomers in the M- (mobile) state can beobtained from the NMR spectra and wasdetermined under different experimental conditions. In thepresence of 150 mM KCl approximately halfof the protomers in Mg-F-actin are in the M-state. This numberis largely independent of the pH in therange studied (pH 7.2-7.8) and of the temperature in range studied(283-310 K). The equilibrium constantKMI for the equilibrium between the I- andM-states is approximately 1.3 under these conditions.