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Conformational changes of the H + ‐ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence
Author(s) -
Börsch Michael,
Turina Paola,
Eggeling Christian,
Fries Joachim R,
Seidel Claus A.M,
Labahn Andreas,
Gräber Peter
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01247-2
Subject(s) - chemistry , fluorescence correlation spectroscopy , fluorescence , nucleotide , molecule , diffusion , fluorescence spectroscopy , confocal , spectroscopy , analytical chemistry (journal) , biophysics , crystallography , biochemistry , biology , chromatography , physics , geometry , mathematics , organic chemistry , quantum mechanics , gene , thermodynamics
Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F 1 part of the H + ‐ATPase from Escherichia coli , EF 1 , carrying the γT106‐C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355–21359] in aqueous solution upon excitation with a mode‐locked argon ion laser at 528 nm. The diffusion of the labeled EF 1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3×10 −7 cm 2 s −1 . In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF 1 molecules allows the detection of conformational changes.