Heintz, D., Kany, H. und Kalbitzer, Hans Robert
(1996)
Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by ¹H and ¹⁹F nuclear magnetic resonance spectroscopy.
Biochemistry 35 (39), S. 12686-12693.
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Zusammenfassung
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 ...
Zusammenfassung
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.
Bibliographische Daten exportieren
| Dokumentenart: | Artikel |
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| Datum: | 1996 |
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| Institutionen: | Biologie und Vorklinische Medizin > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Dr. Hans Robert Kalbitzer |
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| Identifikationsnummer: | | Wert | Typ |
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| 8841112 | PubMed-ID | | 10.1021/bi961159k | DOI |
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| Klassifikation: | | Notation | Art |
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| Acetamides/metabolism | MESH | | Actins/metabolism | MESH | | Amino Acid Sequence | MESH | | Animals | MESH | | Calcium/metabolism | MESH | | Cysteine/chemistry | MESH | | Hydrogen Bonding | MESH | | Hydrogen-Ion Concentration | MESH | | Immunoglobulin Fab Fragments/metabolism | MESH | | Magnesium/metabolism | MESH | | Magnetic Resonance Spectroscopy | MESH | | Models, Molecular | MESH | | Molecular Sequence Data | MESH | | Molecular Structure | MESH | | Muscle, Skeletal/chemistry | MESH | | Protein Conformation | MESH | | Protein Structure, Secondary | MESH | | Rabbits | MESH | | Temperature | MESH |
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| Dewey-Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie |
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| Status: | Veröffentlicht |
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| Begutachtet: | Unbekannt / Keine Angabe |
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| An der Universität Regensburg entstanden: | Unbekannt / Keine Angabe |
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| Eingebracht am: | 13 Sep 2010 08:21 |
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| Zuletzt geändert: | 08 Mrz 2017 08:27 |
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| Dokumenten-ID: | 16547 |
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