Conformation of Contractile Protein in its Transition State as Studied by Phosphorus-31 Nuclear Magnetic Resonance

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Myosin is a contractile protein of muscles and plays an important role in the energy transduction of chemical to physical energy. In the present study, I have studied the conformation of the protein and its interaction with substrate analogs by means of Nuclear Magnetic Resonance (NMR) since NMR gives us a good probe for microenvironments and interactions of molecules.Myosin subfragment 1 (S1) was prepared by the digestion of rabbit skeletal muscle myosin with chymotrypsin and was confirmed by measuring the ratio of Ca-ATPase to Mg-ATPase activity. The ^<31> NMR signal of any complex of myosin S1 and nucleotide was not observed during the ATPase reaction of myosin S1 in NMR sample tube. The NMR signal of the nucleotide-S1 complex might be observed with chicken gizzard myosin because the Mg-ATPase activity of gizzard myosin is much lower than that of rabbit skeletal muscle myosin. We will continue to try to observe the NMR signals of the nucleotide-myosin complex in its transition state of ATPase reaction.The complex formation of ADP and rabbit skeletal muscle myosin S1 was studied at the various temperatures between 0 and 25 ﾟC. The ^<31>P NMR signal of the ADP-S1 complex was obserred at -2.0 ppm. When the temperature was lowered the complex signal reduced its intensity with out broadening and was not observed at 5 ﾟC. This may be due to that myosin S1 has two conformations in S1-nucleotide complex, i.e. high-temperature form and low-temperature form. In the low-temperature form, the amino acid residues of S1 may interact strongly with phosphate moieties of ADP to make electron distribution anisotropy, while electrons of phosphates may be rather isotropic in high-temperatrue form.