Ž . Chemical Physics 247 1999 107–118 www.elsevier.nlrlocaterchemphys Temporal fluctuations of fluorescence resonance energy transfer between two dyes conjugated to a single protein Taekjip Ha a, ) , Alice Y. Ting b , Joy Liang a , Ashok A. Deniz, Daniel S. Chemla a,c , Peter G. Schultz b,d , Shimon Weiss a,d a Materials Sciences DiÕision, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA b Howard Hughes Medical Institute, Department of Chemistry, UniÕersity of California at Berkeley, Berkeley, CA 94720, USA c Department of Physics, UniÕersity of California at Berkeley, Berkeley, CA 94720, USA d Physical Biosciences DiÕision, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Received 1 April 1999 Abstract 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 w attached to a single protein – staphylococcal nuclease molecules T. Ha, A.Y. Ting, J. Liang, W.B. Caldwell, A.A. Deniz, Ž . x D.S. Chemla, P.G. Schultz, S. Weiss, Proc. Natl. Acad. Sci. USA 96 1999 893–898 . Temporal fluctuations in the energy Ž. Ž. Ž. transfer signal include: 1 reversible transitions to dark states; 2 irreversible photodestruction; 3 intersystem crossing to Ž. Ž. Ž. 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 Ž.Ž. 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 dye’s 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 Ž . of the active-site inhibitor deoxythymidine diphosphate to the protein. The inhibitor most probably affects the protein’s stability and the dye–protein interaction, possibly by amplifying the motion of the linker arm between the fluorophore and the protein. q 1999 Elsevier Science B.V. All rights reserved. 1. Introduction Ž . The term single-molecule spectroscopy SMS has been recently used to designate the investigation of ) Corresponding author. Present address: Department of Physics, Stanford University, Stanford, CA 94305, USA. Ž . optical usually fluorescence properties of individ- ual molecules. Because only one molecule is studied at a time, SMS is free from ensemble averaging and allows one to test and compare microscopic theories with experiments. SMS studies of recent years have taught us that fluorescence properties of single molecules are very sensitive to the immediate local 0301-0104r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0301-0104 99 00149-4