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Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.

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Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1–10 nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs.

Theory without experiment is like the sound of one hand clapping. F€ orster theory is not like that at all. It is a thundering ovation linking theory and experiment by explaining the relationship between spectral overlap, energy transfer, and proximity. This chapter explains F€ orster's contributions to the theory of resonance energy transfer. The readers of this chapter form, no doubt, a highly diverse group of people. Most readers are probably only interested in the bottom line. Others may want to know details. But which details? There are so many. To help students and specialists find what they need, the chapter is presented as a sequence of a large number of sections that are short and focused. Pre-F€ orsterThis section is based on some of the information in the most popular papers by F€ orster [1][2][3][4][5], Chapter 5 of Ref. [6], and Clegg's history of FRET [7]. The emphasis here is on the contributions of F€ orster's predecessors and contemporaries. If you want to know who the scientists were who inspired F€ orster and what the science was that motivated him, you should read his most important papers. His most important, that is, his most cited papers are his papers published in 1946 [1] 1948 [2], and 1949 [4] and reviews published in 1959 [3] and 1965 [5]. F€ orster's papers are not easy to understand. The language is not a problem because four of the five are in English or translated into English. They are difficult because they use a lot of math and complicated spectroscopic concepts. Nevertheless, if you are serious about FRET, you should study them. Start with his 1946 paper [1] and the 1959 review [3]. These papers are much more readable than F€ orster's most cited paper [2], because his 1946 paper presents a very clear verbal description of the essential ideas on which the theory is based and a thorough review of the experimental evidence of the importance of resonance and his 1959 paper is designed to provide a conceptual understanding of the FRET -Förster Resonance Energy Transfer: From Theory to Applications, First Edition. Edited by Igor Medintz and Niko Hildebrandt.

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Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.