Single-Molecule FRET (2001)

Single molecule studies have revolutionized our understanding of how biomolecules work. The ability to watch one molecule at a time reveals not only the average properties detected in ensemble measurements, but can yield the entire distribution of relevant properties, including subpopulations and rare events. Single molecule studies also reveal the time evolution of biochemical reactions,which, if asynchronous, are not observable via ensemble measurements. In the 1970s, for example, the development of the patch-clamp technique enabled the electrical current through a single ion channel to be measured, revealing many properties, including that ion channels are on/off devices. More recently, single molecule techniques applicable beyond ion channels have been developed. Methods such as optical (or magnetic) tweezers and scanning force microscopy are making it possible to follow, in real-time, the movements, forces and strains that develop during the course of a reaction (reviewed in [1]). These methods can be used to measure directly the forces that hold together molecular structures. New developments in single molecule fluorescence methodology are also broadening the scope of single molecule studies. Single biomolecules are labeled with an extrinsic fluorophore, and the fluorescent properties of the fluorophore can report on conformational changes occurring in the biomolecules. For example, intensity fluctuations of a single or small number of molecules can also measure reaction rates as well as the number of molecules present. Single enzyme reaction kinetics, for example, were measured in this way and found to have both static and dynamic disorder, as well as a rate which depended on the enzyme's history, properties which were undetectable via ensemble methods [2]. In addition, single molecule fluorescence polarization can be used to infer local rotation or changes in flexibility of biomolecules (reviewed in [3, 4]). Perhaps the most general approach is the use of two fluorophores rather than one, in the form of fluorescence resonance energy transfer (FRET). In FRET, a “donor” dye transfers energy to an “acceptor” dye, and the donor intensity decreases and the acceptor intensity increases as the two dyes approach each other (reviewed in [5]). Importantly, it is sensitive to sub-nanometer distance changes in the range of 20-80 Å, a sensitivity and range well suited for studying biomolecules. Excitation energy of the donor is transferred to the acceptor via an induced-dipole, induced-dipole interaction. The efficiency of energy transfer, E, is given by: where R is the distance between the donor and acceptor and R0 is the distance at which 50% of the energy is transferred and is a function of the properties of the dyes. Because of this strong distance dependence, structural changes of biomolecules or relative motion and interaction between two different molecules can be detected via FRET. These conformational changes are often difficult to synchronize or too rare to detect using ensemble FRET studies. Single-molecule FRET (smFRET) overcomes these difficulties, creating new opportunities to probe structural changes of biological molecules during biological events in real time. SmFRET is also relatively insensitive to incomplete labeling of host molecule with donor and acceptor because donor-only species simply show up as an FRET = 0 peak while acceptor-only species are excited only very weakly by the excitation light if the probes are selected with a large spectral separation. Unlike other single-molecule methodologies measuring forces (optical or magnetic tweezers and atomic force microscopy) [1], single-molecule FRET is sensitive to the internal motion or arrangement of host biological molecules in its center of mass frame; therefore it does not suffer from Brownian motion of the whole molecule. In addition, it can be applied to a much broader class of biological systems, because it does not require force-generating molecules. It is especially powerful in capturing rare, transient events and in dissecting complex multi-step processes. Since our first demonstration of single molecule FRET, there have been a number of biological applications (and see review in [4, 6]). Ever expanding list of biological systems that have been studied by single molecule FRET includes DNA rulers [7-9], Staphylococcal nuclease [10], biotin-Streptavidin [11], GCN4 peptides [12], a-tropomyosin [13], S15 binding RNA junction [14], Tetrahymena ribozyme [15], calmodulin [16], and Rep helicase (Ha et al, unpublished data). There are already excellent reviews on the existing single molecule FRET works [4, 6] and a review focused on practical issues regarding smFRET implementation is available [17].