FLUORESCENCE HETEROGENEITY OF TRYPTOPHANS IN NA,K-ATPASE - EVIDENCES FOR TEMPERATURE-DEPENDENT ENERGY-TRANSFER

The intrinsic fluorescence emission kinetics of Na,K-ATPase, a large m embrane protein containing 16 tryptophan residues, was studied by time -resolved techniques. The lifetime distributions recovered by the Maxi mum Entropy Method exhibit a strong dependence on the emission wavelen gth at temperatures between 37 degrees C and - 70 degrees C. From the 'blue' edge of the fluorescence emission spectrum up to the maximum of emission, the lifetime distribution at room temperature is the result of four broad peaks which cover the time range 0.3-7 ns. With increas ing emission wavelength, these peaks move to longer lifetimes and the peak at shorter times are suppressed at the red edge, while the longes t component (6-7 ns) becomes dominant. With decreasing temperature, th e number of lifetime components is reduced for the benefit of the long one. At cryogenic temperatures, the emission decay in the red-edge of the fluorescence spectrum consists of one major slow component (6-7 n s) and a fast one (0.5 ns) associated with a negative pre-exponential term. This is a characteristic feature of an excited-state reaction. T he temperature dependence of this fast component and the fluorescence anisotropy decay at low temperature in the red-edge, indicate that thi s excited state reaction may be accounted for a unidirectional inter-t ryptophan fluorescence energy transfer from 'blue' populations of dono rs to 'red' populations of accepters. This is also illustrated by the time-resolved emission spectra. In the blue edge of the fluorescence e mission spectrum, moreover, the time course of the anisotropy decay su ggests the existence of home-transfer of excitation energy involving ' blue' tryptophan residues. The steady-state anisotropy excitation spec trum in vitrified solvent agrees with this suggestion. These different energy transfer mechanisms may be used as structural probes to detect more accurately conformational changes of the protein elicited by eff ecters and ion binding or release. (C) 1998 Elsevier Science B.V. All rights reserved.