Cross-linking constraints on F-actin structure.
The DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274), and the C terminus region are among the structural elements of monomeric (G-) actin proposed to form the intermonomer interface in F-actin. To test the proximity and interactions of these elements and to provide constraints on models of F-actin structure, cysteine residues were introduced into yeast actin either at residue 41 or 265. These mutations allowed for specific cross-linking of F-actin between C41 and C265, C265 and C374, and C41 and C265 using dibromobimane and disulfide bond formation. The cross-linked products were visualized on SDS-PAGE and by electron microscopy. Model calculations carried out for the cross-linked F-actins revealed that considerable flexibility or displacement of actin residues is required in the disulfide cross-linked segments to fit these filaments into model F-actin structures. The calculated, cross-linked structures showed a better fit to the Holmes rather than the refined Lorenz model of F-actin. It is predicted on the basis of such calculations that image reconstruction of electron micrographs of disulfide cross-linked C41-C374 F-actin should provide a conclusive test of these two similar models of F-actin structure.
Kim
E
E
Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, CA, 90095, USA.
Wriggers
W
W
Phillips
M
M
Kokabi
K
K
Rubenstein
P A
PA
Reisler
E
E
D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D003198:Computer Simulation; D019327:Copper Sulfate / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D018583:Pliability; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D015003:Yeasts / Q000737:chemistry*
D000199:Actins; D001704:Biopolymers; D001643:Bridged Bicyclo Compounds; D003432:Cross-Linking Reagents; D004220:Disulfides; C037094:dibromobimane; D003545:Cysteine; D019327:Copper Sulfate