Temporal fluctuations of fluorescence resonance energy transfer between two dyes conjugated to a single protein

Biological molecules together with available labeling chemistries provide a n ideal setting to investigate the interaction between two closely spaced d ye molecules. The photo-excitation of a donor molecule can be non-radiative ly transferred to a near-by acceptor molecule via the induced-dipole-induce d-dipole interaction in a distance-dependent manner. In this work, we furth er elaborate on single-molecule fluorescence resonance energy transfer meas urements between two dye molecules attached to a single protein - staphyloc occal nuclease molecules [T. Ha, A.Y. Ting, J. Liang, W.B. Caldwell, A.A. D eniz, D.S. Chemla, P.G. Schultz, S. Weiss, Proc. Natl. Acad. Sci. USA 96 (1 999) 893-898]. Temporal fluctuations in the energy transfer signal include: (1) reversible transitions to dark states; (2) irreversible photodestructi on; (3) intersystem crossing to and from the triplet state; (4) spectral fl uctuations; (5) rotational dynamics of the dyes; and (6) distance changes b etween the two dyes. To extract biologically relevant information from such measurements, an experimental strategy and data analysis schemes are devel oped. First, abrupt photophysical events, such as (1)-(3) are identified an d removed from the data. The remaining slow, gradual fluctuations in the en ergy transfer signal are due to spectral shifts, rotational dynamics and di stance changes of the dyes. Direct measurements of each dye's spectral fluc tuation and rotational dynamics indicate that these, by themselves, cannot fully account for the observed energy transfer fluctuations. It is therefor e concluded that inter-dye distance changes must be present as well. The di stance and orientational dynamics are shown to be dependent on the binding of the active-site inhibitor (deoxythymidine diphosphate) to the protein. T he inhibitor most probably affects the protein's stability and the dye-prot ein interaction, possibly by amplifying the motion of the linker arm betwee n the fluorophore and the protein. (C) 1999 Elsevier Science B.V, All right s reserved.