Ensemble Fluorescence Resonance Energy Transfer Research Articles

Periodic Operation of a Dynamic DNA Origami Structure Utilizing the Hydrophilic-Hydrophobic Phase-Transition of Stimulus-Sensitive Polypeptides.

Dynamic DNA nanodevices are designed to perform structure-encoded motion actuated by a variety of different physicochemical stimuli. In this context, hybrid devices utilizing other components than DNA have the potential to considerably expand the library of functionalities. Here, the reversible reconfiguration of a DNA origami structure using the stimulus sensitivity of elastin-like polypeptides is reported. To this end, a rectangular sheet made using the DNA origami technique is functionalized with these peptides and by applying changes in salt concentration the hydrophilic-hydrophobic phase transition of these peptides actuate the folding of the structure. The on-demand and reversible switching of the rectangle is driven by externally imposed temperature oscillations and appears at specific transition temperatures. Using transmission electron microscopy, it is shown that the structure exhibits distinct conformational states with different occupation probabilities, which are dependent on structure-intrinsic parameters such as the local number and the arrangement of the peptides on the rectangle. It is also shown through ensemble fluorescence resonance energy transfer spectroscopy that the transition temperature and thus the thermodynamics of the rectangle-peptide system depends on the stimuli salt concentration and temperature, as well as on the intrinsic parameters.

Interactions between a Bioflavonoid and c-MYC Promoter G-Quadruplex DNA: Ensemble and Single-Molecule Investigations.

Small molecules capable of stabilizing the G-quadruplex structure of the nuclease hypersensitivity element III1 (NHE III1) are useful in controlling the overexpression of the c-MYC oncogene. In this study, we have probed the interactions of a 22-mer c-MYC promoter quadruplex-forming sequence (Pu22) with a bioflavonoid 3,4',5,7-tetrahydroxyflavone, commonly known as kaempferol (KF). Ensemble fluorescence resonance energy transfer experiments on labeled Pu22 indicate that KF decreases the affinity of the former toward its complimentary strand, suggesting the stabilization of the quadruplex structure of Pu22. Considering that binding dynamics plays an important role in supramolecular interactions, there is hardly any information on this aspect for quadruplex-flavonoid systems; we have studied the kinetics of KF-Pu22 complexation and decomplexation processes on the single-molecule level by employing fluorescence correlation spectroscopy technique. The binding dynamics is characterized by a fast relaxation time of 10-50 μs. This leads to a high association rate constant ( k+) of ∼109 M-1 s-1, which is close to the pure diffusion controlled limit. However, it is the low dissociation rate constant ( k-) of ∼104 s-1 that is mainly responsible for the stability of the KF-Pu22 complex. Molecular docking study shows that KF binds near the 3'-end of Pu22 by forming several H-bonds with the bases. These findings suggest that KF is a potential binder of the c-MYC promoter quadruplex DNA and can be useful in anticancer therapies.

Detection of Mg2+-dependent, coaxial stacking rearrangements in a bulged three-way DNA junction by single-molecule FRET

Three-way helical junctions (3WJs) arise in genetic processing, and they have architectural and functional roles in structured nucleic acids. An internal bulge at the junction core allows the helical domains to become oriented into two possible, coaxially stacked conformers. Here, the helical stacking arrangements for a series of bulged, DNA 3WJs were examined using ensemble fluorescence resonance energy transfer (FRET) and single-molecule FRET (smFRET) approaches. The 3WJs varied according to the GC content and sequence of the junction core as well as the pyrimidine content of the internal bulge. Mg2+ titration experiments by ensemble FRET show that both stacking conformations have similar Mg2+ requirements for folding. Strikingly, smFRET experiments reveal that a specific junction sequence can populate both conformers and that this junction undergoes continual interconversion between the two stacked conformers. These findings will support the development of folding principles for the rational design of functional DNA nanostructures.

Cholesterol-induced lipophobic interaction between transmembrane helices using ensemble and single-molecule fluorescence resonance energy transfer.

The solvent environment regulates the conformational dynamics and functions of solvated proteins. In cell membranes, cholesterol, a major eukaryotic lipid, can markedly modulate protein dynamics. To investigate the nonspecific effects of cholesterol on the dynamics and stability of helical membrane proteins, we monitored association-dissociation dynamics on the antiparallel dimer formation of two simple transmembrane helices (AALALAA)3 with single-molecule fluorescence resonance energy transfer (FRET) using Cy3B- and Cy5-labeled helices in lipid vesicles (time resolution of 17 ms). The incorporation of 30 mol % cholesterol into phosphatidylcholine bilayers significantly stabilized the helix dimer with average lifetimes of 450-170 ms in 20-35 °C. Ensemble FRET measurements performed at 15-55 °C confirmed the cholesterol-induced stabilization of the dimer (at 25 °C, ΔΔG(a) = -9 kJ mol(-1) and ΔΔHa = -60 kJ mol(-1)), most of which originated from "lipophobic" interactions by reducing helix-lipid contacts and the lateral pressure in the hydrocarbon core region. The temperature dependence of the dissociation process (activation energy of 48 kJ) was explained by the Kramers-type frictional barrier in membranes without assuming an enthalpically unfavorable transition state. In addition to these observations, cholesterol-induced tilting of the helices, a positive ΔC(p(a)), and slower dimer formation compared with the random collision rate were consistent with a hypothetical model in which cholesterol stabilizes the helix dimer into an hourglass shape to relieve the lateral pressure. Thus, the liposomal single-molecule approach highlighted the significance of the cholesterol-induced basal force for interhelical interactions, which will aid discussions of complex protein-membrane systems.

Fret-Based Approach to Probe Domain Motions upon ProRS/YbaK/tRNA_Pro Ternary Complex Formation

To obtain a high level of accuracy during protein synthesis, several different quality control steps are employed by the cellular machinery. The aminoacyl-tRNA synthetases (aaRS) play a critical role in identifying amino acids and pairing them with their cognate tRNAs. Prolyl-tRNA synthetase (ProRS) from all three domains of life has been shown to mischarge alanine and cysteine onto tRNAPro. Most bacterial ProRSs have an editing domain that deacylates mischarged Ala-tRNAPro. However, this double-sieve editing mechanism is not sufficient to clear Cys-tRNAPro. Instead, a free-standing homolog of the ProRS editing domain called YbaK deacylates mischarged Cys-tRNAPro species.We have demonstrated that tRNAPro, ProRS and YbaK form a ternary complex in vitro and in vivo, but the details of this complex are not known.Based on preliminary computational studies, we hypothesize that the alanine editing domain of ProRS undergoes a conformational change to facilitate YbaK binding. In addition, the CCA-3′ end of the tRNA must also be involved in significant conformational changes, translocating between the synthetic active site, the ProRS editing domain active site located 35 A away, and YbaK. To probe these protein and RNA domain movements, we devised a fluorescence resonance energy transfer (FRET)-based approach. To date, using ensemble time-resolved FRET, we have measured an ∼20 A conformational change in the ProRS editing domain upon YbaK binding, confirming our hypothesis of a large conformational change to accommodate YbaK. Moreover, the distance between tRNA and YbaK differ by ∼15 A in the presence and absence of ProRS, further verifying a conformational change. Current studies are aimed at obtaining additional distance constraints between components of the ternary complex.

Dynamic Ligand-induced Conformational Rearrangements in P-glycoprotein as Probed by Fluorescence Resonance Energy Transfer Spectroscopy

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anticancer chemotherapy. Here, we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two nucleotide binding domains (NBDs) are undergoing during the catalytic cycle. Pairs of cysteines were introduced into equivalent regions in the N- and C-terminal NBDs for labeling with fluorescent dyes for ensemble and single-molecule FRET spectroscopy. In the ensemble FRET, a decrease of the donor to acceptor (D/A) ratio was observed upon addition of drug and ATP. Vanadate trapping further decreased the D/A ratio, indicating close association of the two NBDs. One of the cysteine mutants was further analyzed using confocal single-molecule FRET spectroscopy. Single Pgp molecules showed fast fluctuations of the FRET efficiencies, indicating movements of the NBDs on a time scale of 10-100 ms. Populations of low, medium, and high FRET efficiencies were observed during drug-stimulated MgATP hydrolysis, suggesting the presence of at least three major conformations of the NBDs during catalysis. Under conditions of vanadate trapping, most molecules displayed high FRET efficiency states, whereas with cyclosporin, more molecules showed low FRET efficiency. Different dwell times of the FRET states were found for the distinct biochemical conditions, with the fastest movements during active turnover. The FRET spectroscopy observations are discussed in context of a model of the catalytic mechanism of Pgp.

Plasticity of the Asialoglycoprotein Receptor Deciphered by Ensemble FRET and Single-Molecule Counting PALM

The composition of many multi-subunited receptor complexes at the plasma membrane is still debated as well as the impact of changes in the receptor subunit composition. Receptor plasticity, involving changes in stoichiometry and receptor function, has been difficult to address. Here, we introduce new spectroscopic tools to dissect receptor subunit assembly of the Asialoglycoprotein Receptor. Using this highly tractable receptor model system we show that the Asialoglycoprotein Receptor can assemble into distinct oligomers at the plasma membrane and that differential subunit assembly dictates receptor specificity. With ensemble analyses of Fluorescence Resonance Energy Transfer (FRET) and analytical modeling, we quantified receptor subunit homo- and hetero-oligomerization in the living cell. Furthermore, we established single-molecule counting using Photoactivated Localization Microscopy (PALM), visualizing an asymmetric receptor subunit assembly on the single-molecule level. Our results define a probability hierarchy of oligomerization driven by different molecular motifs that entails distinct co-existent receptor subunit assemblies. The Asialoglycoprotein Receptor is involved in the clearance of thrombogenic material. The variety of potential ligands and the propensity of the subunits to form distinct oligomers may explain previous inconsistent results and underscore the importance of deciphering oligomerization in the single cell and on the single-molecule level.

Single-molecule FRET study of SNARE-mediated membrane fusion.

Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.

Retroviral Integrases Promote Fraying of Viral DNA Ends

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.

Dynamics of P-Glycoprotein: A Fluorescence and Disulfide Cross-Linking Approach

P-glycoprotein (Pgp), a member of the ABC transporter family, couples ATP hydrolysis to efflux of hydrophobic molecules including drugs used in chemotherapy. Here we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two NBDs are undergoing. Cysteines introduced into equivalent regions in the N- and C-terminal NBDs of cysteine-less mouse Pgp were labeled with fluorescent dyes for ensemble and single molecule FRET (smFRET) spectroscopy with lipid-reconstituted Pgp. In ensemble fluorescence experiments, adding substrate and/or nucleotide increased the FRET efficiency for all mutants to varying degrees, suggesting that the two NBDs approach one another upon substrate binding. Analysis of smFRET data suggests that the NBDs of Pgp alternate between at least two major conformations, a low and a high FRET state, during verapamil stimulated ATP hydrolysis. Vanadate inhibition shifted the population toward the high FRET state while the Pgp inhibitor cyclosporin resulted in a shift towards the low FRET state. Furthermore, we have utilized direct disulfide cross-linking to test whether complete dissociation of the NBDs is required for ATPase stimulation by bulky drug molecules. Tethering the two NBDs together at their C-terminal ends did not abolish drug stimulated ATP activity, indicating the motion the NBDs are undergoing is mainly on the level of the catalytic sites, with the C-terminal ends of the NBDs acting as a hinge. Taken together, the data provide an indication as to what the nature and magnitude of the structural rearrangements are that Pgp is undergoing during the catalytic cycle.

Structure of the three-way helical junction of the hepatitis C virus IRES element

The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS(4) junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS(1)HS(3) and 2HS(2)HS(2) junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg(2+) ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.

Probing the Conformational Distributions of Subpersistence Length DNA

We have measured the bending elasticity of short double-stranded DNA (dsDNA) chains through small-angle x-ray scattering from solutions of dsDNA-linked dimers of gold nanoparticles. This method, which does not require exertion of external forces or binding to a substrate, reports on the equilibrium distribution of bending fluctuations, not just an average value (as in ensemble fluorescence resonance energy transfer) or an extreme value (as in cyclization), and in principle provides a more robust data set for assessing the suitability of theoretical models. Our experimental results for dsDNA comprising 42–94 basepairs are consistent with a simple wormlike chain model of dsDNA elasticity, whose behavior we have determined from Monte Carlo simulations that explicitly represent nanoparticles and their alkane tethers. A persistence length of 50 nm (150 basepairs) gave a favorable comparison, consistent with the results of single-molecule force-extension experiments on much longer dsDNA chains, but in contrast to recent suggestions of enhanced flexibility at these length scales.

Monitoring Protein Conformation along the Pathway of Chaperonin-Assisted Folding

The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.

Site-directed Mutations of T4 Helicase Loading Protein (gp59) Reveal Multiple Modes of DNA Polymerase Inhibition and the Mechanism of Unlocking by gp41 Helicase

The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.

Metal Ion Dependence, Thermodynamics, and Kinetics for Intramolecular Docking of a GAAA Tetraloop and Receptor Connected by a Flexible Linker

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] << [Ca(2+)], [Mg(2+)], [Mn(2+)] << [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.

Single-Molecule FRET

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 A, 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].

Single-Molecule FRET

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].