title: 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.
abstract: We have synthesized the luminescent and fluorescent lanthanide chelate S-(2-nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-aminoethyl)am ino ]naphthalene-1-sulfonic acid as well as the fluorescent analogue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancourt, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disulfides react with the skeletal myosin motor domain (S-1) as actin site-directed agents and label exclusively and stoichiometrically Cys 540 in the hydrophobic 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 cysteaminyl probe side chain spans maximally 17 and 8 A, respectively, and the fluorophores have different polarity, volume, and flexibility. Thus, they may provide complementary spectroscopic information on the environmental properties of this critical actin binding region. Here, we have analyzed by extrinsic fluorescence spectroscopy S-1 derivatized with the fluorescein label or with the Tb(3+) or Eu(3+) chelate of the other label to assess the conformational transitions precisely occurring at this site upon interaction with F-actin, nucleotides, or phosphate analogues. For either label, specific spectral changes of significant amplitude were obtained, identifying at least two major structural states. One was mediated by rigor binding of F-actin in the absence or presence of MgADP. It was abolished by MgATP, and it was not produced by the binding of nonpolymerizable G-actin. A modeling of the corresponding changes in the intensity and lambda(max) of the fluorescence emission spectra, achieved using the fluorescent adducts of 2-mercaptoethanol in varying concentrations of dimethylformamide, illustrates the 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 PP(i). It should be prevalent in the weak acto-S-1 binding complexes. The accompanying 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 spectral 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 affinity. Our study offers the basis necessary for further detailed spectroscopic investigations on the conformational dynamics in solution of the stereospecific and hydrophobic actin binding motif during the skeletal cross-bridge cycle.
authors.affiliation: Centre de Recherches de Biochimie Macromoléculaire du CNRS, UPR 1086, 1919 Route de Mende, 34293 Montpellier Cedex 5, France.
authors.forename: R
authors.initials: R
authors.lastname: Bertrand
authors.forename: J
authors.initials: J
authors.lastname: Derancourt
authors.forename: R
authors.initials: R
authors.lastname: Kassab
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D046911:Macromolecular Substances; D018482:Muscle, Skeletal / Q000378:metabolism*; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009281:Naphthalenes / Q000378:metabolism; D009579:Nitrobenzoates / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence
chemical_list: D000199:Actins; D004220:Disulfides; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D015879:Myosin Subfragments; C418055:NTB-cysteaminyl-DTPA-EDANS; C418056:NTB-cysteaminylcarboxyfluorescein; D009281:Naphthalenes; D009579:Nitrobenzoates; D019920:Photoaffinity Labels; D003545:Cysteine