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

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F₄/^-), fluoroberyllate (BeF(n)) 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. and 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 γ-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. and 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/^- BeF(n) 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 BeF(n) 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 BeF(n) was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeF(n) complex than for AlF₄/^- complex. The ¹⁹F-NMR spectra of the bound 2-[(trifluoromelhylnitrophenyl)-aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF₄/^-, BeF(n) or Vi showed small but distinguishable differences. Taken together, these results indicate that them 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.