Cross-linking constraints on F-actin structure

The DNase I binding loop (residues 38-52), the hydrophobic plug (residues 2 62-274), and the C terminus region are among the structural elements of mon omeric (G-) actin proposed to form the intermonomer interface in F-actin. T o test the proximity and interactions of these elements and to provide cons traints on models of F-actin structure, cysteine residues were introduced i nto yeast actin either at residue 41 or 265. These mutations allowed for sp ecific cross-linking of F-actin between C41 and C265, C265 and C374, and C4 1 and C265 using dibromobimane and disulfide bond formation. The cross-link ed products were visualized on SDS-PAGE and by electron microscopy. Model c alculations carried out for the cross-linked F-actins revealed that conside rable flexibility or displacement of actin residues is required in the disu lfide cross-linked segments to fit these filaments into model F-actin struc tures. The calculated, cross-linked structures showed a better fit to the H olmes rather than the refined Lorenz model of F-actin. It is predicted on t he basis of such calculations that image reconstruction of electron microgr aphs of disulfide cross-linked C41-C374 F-actin should provide a conclusive test of these two similar models of F-actin structure. (C) 2000 Academic Press.