Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils

:Myosin subfragment 1 (S1) can be specifically modified at Lys-553 with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid s uccinimidyl ester) (Bertrand, R,, J. Derancourt, and R. Kassab. 1995. Bioch emistry. 34.9500-9507), and solvent quenching of FHS-S1 with iodide has bee n shown to be sensitive to actin binding at low ionic strength (MacLean, Ch rin, and Berger, 2000. Biophys, J. 000- 000). In order to extend these resu lts and examine the fraction of actin-bound myosin heads within the myofila ment lattice during calcium activation, we have modified skeletal muscle my ofibrils, mildly cross-linked with EDC (1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide) to prevent shortening, with FHS, The myosin heavy chain appea rs to be the predominant site of labeling, and the iodide quenching pattern s are consistent with those obtained for myosin SI in solution, suggesting that Lys-553 is indeed the primary site of FHS incorporation in skeletal mu scle myofibrils. The iodide quenching results from calcium-activated FHS-my ofibrils indicate that during isometric contraction 29% of the myosin heads are strongly bound to actin within the myofilament lattice at low ionic st rength., These results suggest that myosin can be specifically modified wit h FHS in more complex and physiologically relevant preparations, allowing t he real time examination of cross-bridge interactions with actin in in vitr o motility assays and during isometric and isotonic contractions within sin gle muscle fibers.