The structural changes in enzymes that accompany substrate binding, reaction and product release are generally on a scale of 1-2A; in muscle, ATP hydrolysis induces structural changes on a scale of 50A per crossbridge cycle. To investigate the mechanism by which large-scale structural change is generated and regulated, we will determine the spatial relations among structural domains and key functional sites in the actin-myosin complex, using as our primary methods site-specific labeling with gold cluster reagents, high-resolution scanning transmission electron microscopy and computational image analysis. In actin, we will determine the location of a reactive glutamine residue; using its position and the position of cysteine-374 as reference points, we will orient the monomer in the actin filament. In the myosin head, we will determine the locations of the intrinsic reactive thiol groups of the heavy chain (cysteine-707, or SH-1) and alkali light chains. We will label reactive lysine and glutamine residues in both light and heavy chains, and thereby determine both the positions of the light chains within the myosin head and the positions of the tryptic domains of the heavy chain. In collaboration with Dr. Ralph Yount, we will determine the position of undecagold-nucleotide bound at the active site. Gold cluster labeling offers the unique opportunity to apply a new scattering center to a biochemically-defined site, determine its position in real space by electron microscopy, and observe its behavior in the wet state by X-ray methods. We will test various strategies for incorporating gold cluster labels at specific sites on myosin heads in muscle fibers. In collaboration with Dr. Carolyn Cohen, we will determine the geometry of labeled myosin heads bound to actin in the presence and absence of nucleotide. We will also investigate the use of solutions of labeled molecules as X-ray specimens.
| Safer, D (1999) Undecagold cluster labeling of proteins at reactive cysteine residues. J Struct Biol 127:101-5 |
| Safer, D; Golla, R; Nachmias, V T (1990) Isolation of a 5-kilodalton actin-sequestering peptide from human blood platelets. Proc Natl Acad Sci U S A 87:2536-40 |
| Safer, D (1989) An electrophoretic procedure for detecting proteins that bind actin monomers. Anal Biochem 178:32-7 |
