Fluorescence characterization of structural transitions at the strong actin binding motif in skeletal myosin affinity labeled at cysteine 540 with novel spectroscopic cysteaminyl mixed disulfides

We have synthesized the luminescent and fluorescent lanthanide chelate S-(2 -nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-a minoethyl)amino] naphthalene-1-sulfonic acid as well as the fluorescent ana logue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancour t, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disu lfides react with the skeletal myosin motor domain (S-1) as actin site-dire cted agents and label exclusively and stoichiometrically Cys 540 in the hyd rophobic strong actin binding helix-loop-helix motif, causing only a 1.9-2. 4-fold decrease in the V-max for acto-S-1 ATPase. The covalently attached c ysteaminyl probe side chain spans maximally 17 and 8 Angstrom, respectively , and the fluorophores have different polarity, volume, and flexibility. Th us, they may provide complementary spectroscopic information on the environ mental properties of this critical actin binding region. Here, we have anal yzed by extrinsic fluorescence spectroscopy S-1 derivatized with the fluore scein label or with the Tb3+ Or Eu3+ chelate of the other label to assess t he conformational transitions precisely occurring at this site upon interac tion with F-actin, nucleotides, or phosphate analogues. For either label, s pecific spectral changes of significant amplitude were obtained, identifyin g at least two major structural states. One was mediated by rigor binding o f F-actin in the absence or presence of MgADP. It was abolished by MgATP, a nd it was not produced by the binding of nonpolymerizable G-actin. A modeli ng of the corresponding changes in the intensity and lambda (max) of the fl uorescence emission spectra, achieved using the fluorescent adducts of 2-me rcaptoethanol in varying concentrations of dimethylformamide, illustrates t he predicted apolar nature of the strong acto-S-1 interface. A second state was promoted by the binding of ATP, AMP-PNP, ADP.AlF4, ADP.BeFx, or PPi. I t should be prevalent in the weak acto-S-1 binding complexes. The accompany ing fluorescence intensity reduction, observed with each label, in both the absence and presence of F-actin, would result from a specific modification by these ligands of the probe orientation and/or solvent accessibility as suggested by acrylamide quenching experiments. It could represent the spect ral manifestation of the predicted allosteric linkage from the ATPase site to the strong actin binding site of S-1 that modulates the acto-S-1 affinit y. Our study offers the basis necessary for further detailed spectroscopic investigations on the conformational dynamics in solution of the stereospec ific and hydrophobic actin binding motif during the skeletal cross-bridge c ycle.