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Real-Time Measurement of Myosin–Nucleotide Noncovalent Complexes by Electrospray Ionization Mass Spectrometry

Howard D. White ^* and Alison E. Ashcroft 

^* Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 23507; and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

Correspondence: Address reprint requests to Howard D. White, Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia 2350. E-mail: whitehd{at}evms.edu.

Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 ± 10 Da were obtained with the binding of ADP to the active site and by 530 ± 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of 1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 µM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.

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