ANALYSIS OF STRESS IN THE ACTIVE-SITE OF MYOSIN ACCOMPANIED BY CONFORMATIONAL-CHANGES IN TRANSIENT STATE INTERMEDIATE COMPLEXES USING PHOTOAFFINITY-LABELING AND F-19-NMR SPECTROSCOPY

Myosin forms stable ternary complexes with ADP and the phosphate analo gues, fluoroaluminate (AlF4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the my osin ATPase cycle. Moreover, we previously demonstrated that these com plexes may mimic different myosin ATPase reaction intermediates corres ponding 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) thro ugh protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mec hanism by which energy in ATP is transferred to the myosin structure [ Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-33 72]. In the present study, the photoactive ADP analogue, 3'-O-(N-methy lanthraniloyl)-2-azido-ADP (Mant-2-N-3-ADP), and the F-19-labeled ADP analogue, trifluoromethylnitrophenyl)aminoethyl]diphosphate, were empl oyed to examine conformational differences in protein-nucleotide conta ct in the ATP-binding site that may correlate with energy transduction . Mant-2-N-3-ADP was trapped within the active site of skeletal and sm ooth muscle myosin in the presence of AlF4-, BeFn, or Vi. For both ske letal and smooth muscle myosins, trapped Mant-2-N-3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-of both myosin/Mant-2-N -3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the ef ficiency of the incorporation was much higher for skeletal than for sm ooth muscle myosin suggesting that the conformations of the adenine-bi nding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N-3-ADP trapped in the presence of AlF4- and BeFn, wa s the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn, complex tha n for AlF4- complex. The F-19-NMR spectra of the bound (trifluoromethy lnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn, or Vi showed small but distinguishable d ifferences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base am ong the ternary complexes studied, and these differences mimic separat e steps occurring transiently during the contractile cycle.