Intermolecular dynamics and function in actin filaments.
Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin. To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal alpha-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments. Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved alpha-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38-52 in F-actin. Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 A) in accordance with the models of F-actin structure. The dynamic properties of the intermolecular interface in F-actin formed by loop 38-52, plug 262-274 and the C-terminus may account for the observed cross-linking of these sites with reagents < 18 A. The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41-Cys-374 cross-linked actin filaments.
Kim
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Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Reisler
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D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000378:metabolism*; D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D003432:Cross-Linking Reagents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011721:Pyrenes / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000199:Actins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D011721:Pyrenes