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

Biological molecules together with available labeling chemistries provide an ideal setting to investigate the interaction between two closely spaced dye molecules. The photo-excitation of a donor molecule can be non-radiatively transferred to a near-by acceptor molecule via the induced-dipole–induced-dipole interaction in a distance-dependent manner. In this work, we further elaborate on single-molecule fluorescence resonance energy transfer measurements between two dye molecules attached to a single protein – staphylococcal nuclease molecules [T. Ha, A.Y. Ting, J. Liang, W.B. Caldwell, A.A. Deniz, D.S. Chemla, P.G. Schultz, S. Weiss, Proc. Natl. Acad. Sci. USA 96 (1999) 893–898]. Temporal fluctuations in the energy transfer signal include: (1) reversible transitions to dark states; (2) irreversible photodestruction; (3) intersystem crossing to and from the triplet state; (4) spectral fluctuations; (5) rotational dynamics of the dyes; and (6) distance changes between the two dyes. To extract biologically relevant information from such measurements, an experimental strategy and data analysis schemes are developed. First, abrupt photophysical events, such as (1)–(3) are identified and removed from the data. The remaining slow, gradual fluctuations in the energy transfer signal are due to spectral shifts, rotational dynamics and distance changes of the dyes. Direct measurements of each dyeʹs spectral fluctuation and rotational dynamics indicate that these, by themselves, cannot fully account for the observed energy transfer fluctuations. It is therefore concluded that inter-dye distance changes must be present as well. The distance and orientational dynamics are shown to be dependent on the binding of the active-site inhibitor (deoxythymidine diphosphate) to the protein. The inhibitor most probably affects the proteinʹs stability and the dye–protein interaction, possibly by amplifying the motion of the linker arm between the fluorophore and the protein.