CONFORMATIONAL-CHANGES OF THE H-ATPASE FROM ESCHERICHIA-COLI UPON NUCLEOTIDE-BINDING DETECTED BY SINGLE-MOLECULE FLUORESCENCE()

Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine l abeled F-1 part of the H+-ATPase from Escherichia coli, EF1, carrying the gamma T106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. B iol. Chem. 267, 21355-21359] in aqueous solution upon excitation with a mode-locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which wer e analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3 x 10(-7) cm(2) s(-1). In the presence of nucleotides the diffusion coefficient increases by about 15%. This eff ect indicates a change of the shape and/or the volume of the enzyme up on binding of nucleotides, i.e, fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational chang es. (C) 1998 Federation of European Biochemical Societies.