Cross-link between cys 374 and cys 10 of actin abolishes polymerizability and allows study of the properties of the "F-actin monomer".
Actin cross-linked between cys 374 and cys 10 via a disulfide-containing bridge, c-A, is completely unpolymerizable even in the presence of phalloidin. Upon the addition of dithiothreitol, c-A polymerizes with high yield, indicating that denaturation due to the modification was almost absent. In the present study we show that cross-linked actin is a useful model for studying the properties of monomeric actin under polymerization conditions. Addition of salt, for example, produced fluorescence changes possibly reflecting conformational transitions but did not lead to the development of phalloidin binding capacity. Cross-linking of the two cysteine residues also caused a decrease in the nucleotide exchange rate by a factor of ca. 3, an effect that was fully reversed by the addition of KCl. Cross-linked actin inhibits DNase I to the same extent as G-actin and binds thymosin beta 4 and profilin as shown by cross-linking studies. Capping capacity for the barbed end of the filament was not observed, although it might have been expected from the fact that both ends of the cross-link are anchored to subdomain 1. Using the 61-FITC derivative of c-A we showed that c-A is able to bind to myosin S1 with a KD in the microM range. In agreement with this, c-A shows actomyosin ATPase activity with a Kapp comparable to that of F-actin, but a Vmax decreased by a factor of ca. 11. The c-A myosin S1 complex provides the hitherto smallest model of actomyosin, which appears promising for crystallization and X-ray analysis.
Heintz
D
D
Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany.
Faulstich
H
H
D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D003488:Cyanogen Bromide; D003545:Cysteine; D003850:Deoxyribonuclease I / Q000378:metabolism; D004220:Disulfides; D005033:Ethylmaleimide; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010590:Phalloidine / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000199:Actins; D004220:Disulfides; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010590:Phalloidine; D011189:Potassium Chloride; D003850:Deoxyribonuclease I; D009218:Myosins; D016650:Fluorescein-5-isothiocyanate; D003545:Cysteine; D005033:Ethylmaleimide; D003488:Cyanogen Bromide