title: Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F-NMR spectroscopy.
abstract: Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.
authors.affiliation: Department of Bioengineering, Soka University, Hachioji, Tokyo, Japan. shinsaku@t.soka.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Maruta
authors.forename: G D
authors.initials: GD
authors.lastname: Henry
authors.forename: T
authors.initials: T
authors.lastname: Ohki
authors.forename: T
authors.initials: T
authors.lastname: Kambara
authors.forename: B D
authors.initials: BD
authors.lastname: Sykes
authors.forename: M
authors.initials: M
authors.lastname: Ikebe
mesh_terms: D000345:Affinity Labels; D000535:Aluminum / Q000378:metabolism; D000818:Animals; D001608:Beryllium / Q000378:metabolism; D001665:Binding Sites; D005459:Fluorides / Q000378:metabolism; D005461:Fluorine / Q000378:metabolism; D005895:Gizzard, Avian; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D013314:Stress, Mechanical; D014422:Turkeys; D014638:Vanadates / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D015879:Myosin Subfragments; C053629:fluoroaluminum; D005461:Fluorine; D014638:Vanadates; C035982:beryllium fluoride; D000535:Aluminum; D009218:Myosins; D001608:Beryllium; D005459:Fluorides