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* School of Chemistry, Seoul National University, Seoul, Korea; Department of Chemistry and Biochemistry, and Department of Physiology, University of California, Los Angeles, California; and Clarendon Laboratory, Department of Physics, and IRC in Bionanotechnology, University of Oxford, Oxford, United Kingdom
Correspondence: Address reprint requests to Seong Keun Kim, School of Chemistry, Seoul National University, Seoul 151-747, Korea. Tel.: 82-2-880-6659; Fax: 82-2-889-5719; E-mail: seongkim{at}snu.ac.kr; or to Shimon Weiss, Dept. of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Dr., E. Los Angeles, CA 90095. Tel.: 310-794-0093; Fax: 310-267-4672; E-mail: sweiss{at}chem.ucla.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSION AND OUTLOOKSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSION AND OUTLOOKSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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–3) and single-molecule levels (4–6). Most FRET experiments employ a pair of complementary fluorescent probes, a donor and an acceptor; when the donor and acceptor probes are <8–10 nm apart, the measured FRET efficiency can report a single interprobe distance and a bimolecular interaction (1,6).
Recently, several groups introduced ensemble-based three-color FRET (3c-FRET) approaches, which use molecules labeled with three probes, each having a distinct emission spectrum (Fig. 1, A and B; B (blue probe), G (green probe), and R (red probe)) to monitor three-molecule interactions and up to three interprobe distances (7–18). At the single-molecule level, often the only level for handling extensive compositional or conformational heterogeneity, 3c-FRET has also been realized for surface-immobilized (19) and diffusing molecules (20,21), establishing the potential of 3c-FRET for probing complex dynamics of biomolecules.
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). Moreover, no existing method can detect species present in the interaction equilibrium but not appreciably excited by the single-laser excitation used in these methods (such as species G-R, G-only, and R-only (Fig. 1 B)). Finally, in studies of multiple interprobe distances, the existing methods cannot easily measure accurate FRET from single molecules, especially without auxiliary experiments that measure FRET between individual pairs. These limitations cannot be addressed by ensemble-type 3c-FRET (12,13).
Here, we introduce three-color alternating-laser excitation (3c-ALEX), a general method that overcomes the above limitations by implementing alternating-laser excitation (Fig. 1 C) of single molecules labeled with up to three fluorescent probes. The new method is a substantial extension of two-color (2c)-ALEX (23–25), which employs two alternating lasers to study molecular interactions (through probe stoichiometry ratio S) and intramolecular distances (through FRET efficiency E). In 2c-ALEX, molecules are sorted in a two-dimensional histogram of S and E, whereas 3c-ALEX sorts molecules in three-dimensional stoichiometry and three-dimensional FRET histograms. The stoichiometry histograms allowed identification and selection of molecules according to probe-stoichiometry; selected molecules were subsequently represented in FRET-efficiency histograms, where three intermolecular distances were observed. Using triply labeled DNAs (Fig. 2), we demonstrated that 3c-ALEX allows 1), FRET-independent analysis of three-component interactions; 2), observation and sorting of singly, doubly, and triply labeled molecules present in the same solution; 3), measurements of three intramolecular distances within a species even in the absence of substantial FRET among the probes; and 4), dissection of conformational heterogeneity with improved resolution compared to conventional single-molecule FRET (hereafter, 2c-FRET). We used 3c-ALEX to study large biomolecules such as complexes of RNA polymerase (RNAP) with DNA, and to monitor RNAP translocation on DNA from two perspectives within the complex. Single-molecule analysis of complex mixtures and biomolecular machinery should be greatly improved by using 3c-ALEX.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSION AND OUTLOOKSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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). The ratio of the photon counts due to two individual laser excitations reports on probe stoichiometry S. The two-dimensional E-S histogram allows fluorescence-aided molecule sorting. Similarly, 3c-ALEX can measure E and S simultaneously for a three-probe system (B-G-R, where probe B can serve as a donor to probes G and R, and where probe G can serve as a donor to probe R (Fig. 1 A)) from single diffusing molecules, employing three lasers and three detectors (Fig. 1 C). Each laser excites primarily one of the three probes, and the lasers are modulated faster (120 µs) than the diffusion time of a molecule through the observation volume (
1 ms). The fluorescence emissions of the three probes are subsequently detected using three detectors.
The first laser, at 477 nm, excites primarily probe B for the time domain of 0–36 µs within a 120-µs modulation period (Fig. 1 D, left). The excited-state energy of B may be transferred to G and R via FRET (Fig. 1 A), generating photon counts 
, 
, and 
(which denote cross-talk-corrected photon counts from a single fluorescent molecule excited by a 477-nm laser and detected by B-, G-, and R-detectors, respectively; for details see Supplementary Material, Theory of 3c-ALEX and Fig. S1). The three photon counts are used for calculating the following FRET-related ratios:
 | (1) |
 | (2) |
is a proximity ratio (an approximation of FRET efficiency 
(22)). However, proximity ratios 
and 
(also encountered in single-laser 3c-FRET (20)) cannot determine all possible FRET efficiencies among the three probes within a single molecule. To recover the complete set of FRET efficiencies in the case of single-excitation 3c-FRET, an additional measurement on a doubly labeled species is required (20); this is difficult when simultaneous monitoring of three distances within a single molecule is sought, or when analysis of multi-conformer mixtures is performed. In 3c-ALEX, however, the presence of a second laser allows determination of all three FRET efficiencies (and corresponding distances) from single molecules.
The second laser, at 532 nm, excites primarily probe G for the time domain of 40–76 µs (Fig. 1 D, middle). The excited-state energy of G may be transferred to R via FRET, generating two nonzero photon counts, 
and 
; the two photon counts define proximity ratio 
, the ratio not available in single-excitation 3c-FRET:
 | (3) |
The three proximity ratios yield three corrected FRET efficiencies (
, 
and 
; see Supplementary Material, Theory of 3c-ALEX), which directly report on three distances 
, since
 | (4) |
is the Förster radius of the XY probe pair (3).
As in 2c-ALEX (23), 3c-ALEX can measure the probe stoichiometries of the species formed by a three-component, three-probe system independent of FRET. Since photon counts , , and (Fig. 1 D) are generated by exciting probe B by the 477-nm laser, their sum, 
(
), reports on the presence of probe B in a molecule. If a molecule contains probe B, is significant, whereas if the molecule is devoid of B, is negligible. Likewise, the sum of 532-nm–induced photon counts, 
(
), reports on the presence of probe G in a molecule; and the 633-nm-induced photon count 
(
, collected during the time domain of 80–116 µs (Fig. 1 D)) reports on the presence of probe R in a molecule. Using photon counts , , and , we define:
 | (5) |
 | (6) |
 | (7) |
denotes the probe stoichiometry specific to probes X and Y. Since each species generated by three-component interactions (B-only, G-only, R-only, B-G, B-R, G-R, and B-G-R (Fig. 1 B)) has a distinct set of probe stoichiometries (
, 
, and
(Fig. 3 A, Table)), this set can be used to sort, identify, and select molecules belonging to each species. To sort molecules based on probe stoichiometry, we devised a three-dimensional stoichiometry histogram (hereafter, S histogram (Fig. 3)). We use a similar three-dimensional proximity-ratio histogram to display the three proximity ratios (Eqs. 1–3) that characterize each diffusing molecule (Fig. 4); we refer to this histogram as the "E* histogram".
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). To test the ability of 3c-ALEX to measure distances, we prepared DNA fragments C3-b and C3-c which have a different set of intramolecular distances compared to C3-a. The three probes used were Alexa488 (probe B), carboxytetramethylrhodamine (TMR, probe G), and Alexa647 (probe R) (all from Molecular Probes, Eugene, OR) (23). For single-molecule measurements, the final DNA concentration was 50–75 pM in SM buffer (10 mM HEPES-NaOH pH 7, 500 mM NaCl, 100 µg/ml BSA, 1 mM mercaptoethylamine, and 5% glycerol). The Ro values for the B-G, G-R, and B-R pairs were 62 Å, 62 or 66 Å, and 56 Å, respectively (Supplementary Material, Ensemble measurement).
Preparation of transcription complexes
RNAP labeled at position 496 of 
70 was prepared using minor modifications of published protocols (26). Plasmid pGEMD496C was used for overexpression of a 70 derivative with a single reactive Cys at position 496; the 70 derivative was purified as described (27), but using an anion-exchange Mono Q column (Amersham Pharmacia Biotech, Piscataway, NJ) instead of DE52 resin. RNAP labeling was performed by reacting Cys-496 with Cy3B-maleimide (probe G), followed by purification on a gel-filtration column (Y. Kim and S. Weiss, unpublished), followed by reconstitution of RNAP holoenzyme by incubating Cy3B-labeled 70 with RNAP core (Epicentre, Madison, WI) (26).
The DNA used for studying transcription complexes was derived from positions –39 to +20 of a lacCONS sequence (28), and was prepared as described (26), but using Alexa488- and Alexa647-labeled PCR primers (Fig. 2 B). RNAP-DNA complexes were prepared and analyzed as described (29), except that elongation complex RDe,12 was formed by adding 100 µM NTPs and 500 µM 3-OMe-CTP to RPo.
Single-molecule data acquisition and analysis
A 2c-ALEX single-molecule fluorescence microscope (23) was modified for 3c-ALEX (Fig. 1 C). Three laser excitation sources of 477-nm light from Ar+ laser (35-LAP-321, Melles-Griot, Carlsbad, CA), 532-nm light from solid-state green laser (DPGL-20P, World Star Tech, Toronto, Canada), and 633-nm light from HeNe laser (25-LHP-925, Melles-Griot) were alternated using a combination of polarizers with electrooptical modulators (EOMs) (Conoptics, Danbury, CT) (23). The extinction ratios (ratios of laser intensities when a laser is on or off) were typically >200:1 for each laser. The excitation beams were circularly polarized using 
/4 waveplates, coupled through two dichroic mirrors (DM1 and DM2; 560DRLP and 505DRLP; Omega Optical, Brattleboro, VT), and then spatially filtered through a single-mode fiber (460HP, Thorlabs, Newton, NJ). The coupled lights from the fiber were collimated, directed to an IX71 inverted microscope (Olympus, Tokyo, Japan), reflected on a triple-laser beamsplitter (DM3; Z488-533-633RPC, Chroma, Rockingham, VT), and focused 20 µm from the bottom coverslip through a water-immersion objective (60x, 1.2 NA, UPLAPO, Olympus). Fluorescence was collected through the objective and DM3, focused in a 100-µm pinhole, and refocused using a biconvex lens onto silicon avalanche photodiode detectors (APD) (SPCM AQR-14, EG&G, Perkin Elmer, Wellesley, MA) that were connected to a counting board (PCI-6602, National Instruments, Austin, TX). After the biconvex lens fluorescence was separated into three streams by two beamsplitters (DM4 and DM5; 560DRLP and 660DRLP, Omega Optical) and filtered in front of each APD (for Alexa 488, F1, HQ510/30; for TMR, F2, HQ600/40; for Alexa 647, F3, HQ665LP; from Chroma, McHenry, IL). The excitation intensities measured before DM3 were 60–70 µW for 477 nm, 90–120 µW for 532 nm, and 24 µW for 633 nm in alternating mode. This set of filters, beamsplitters, and excitation wavelengths reduced cross talks and background noise efficiently; the contributions of cross talks (summarized in Supplementary Material, Table S1) were <20% of main signals, and the background noises, measured using SM buffer and subtracted for data analysis, were <0.7 kHz for each detection channel. Data analysis was performed using LabVIEW (National Instruments) (24). From the 600-µs-binned photon time traces, we selected molecules using a start/stop criterion on the sum of all photon counts (for time traces, see Supplementary Material, Fig. S2). Each molecule was assigned with nine photon counts 
; six photon counts (in fact, three photon counts of 
, 
, and 
are zero) were used for calculating probe stoichiometries (Eqs. 5–7) and proximity ratios (Eqs. 1–3). After molecular identification, we 1), plotted S histograms; 2), graphically selected species of interest; 3), plotted E* histograms for selected species and fitted them to Gaussian functions to recover mean ; 4), calculated three FRET efficiencies ; and 5), determined three distances (Eq. 4).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSION AND OUTLOOKSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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We first examined three singly labeled species (B-only, G-only, and R-only), and plotted the S histogram for each species in Fig. 3 B. In S histograms, each species appeared as a cluster of points in three-dimensional space; the location of each cluster agreed well with theoretical predictions (Fig. 3 A). To test whether 3c-ALEX can sort and count each singly labeled species in a mixture, we examined an equimolar mixture of all three species. The three species clustered at different locations of the S histogram (Fig. 3 D); the locations matched the locations seen for pure singly labeled species (Fig. 3 B). To examine whether 3c-ALEX can quantify the relative abundance of each species in the mixture, we graphically selected and counted molecules for each sorted singly labeled species (Fig. 3 D, ovals). We counted 404, 452, and 462 molecules of B-only, G-only, and R-only species, respectively, in agreement with the equimolar mixing ratio.
Second, we examined three doubly labeled species (B-G, G-R, and B-R), and plotted S histograms for each species (Fig. 3 C). Again, each species appeared as a cluster of points at the predicted locations (with traces of singly labeled species due to incomplete labeling and inactive states of probes (22)). To test whether 3c-ALEX can sort and count each doubly labeled species in a mixture, we again examined an equimolar mixture of the three doubly labeled species (Fig. 3 E) and obtained clusters matching the ones seen for pure doubly labeled species (Fig. 3 C). The molecular count for the B-G, G-R, and B-R species was 460, 386, and 445, respectively, in good agreement with the equimolar mixing ratio.
Third, we studied a mixture containing triply labeled species B-G-R (Fig. 3 F). A comparison of Fig. 3 F with Fig. 3 E (in which all fluorescent species except B-G-R are present) reveals a new species at the center of the histogram in Fig. 3 F. The new species corresponds to B-G-R, for which all three probe stoichiometries are 0.5 (Fig. 3 A). Since the triply labeled species is clearly isolated from singly and doubly labeled species in the S histogram, the triply labeled species can be easily identified and selected for further analysis.
Our results establish that 3c-ALEX permits sensitive detection, sorting, identification, and molecular counting of singly, doubly, and triply labeled species in complex mixtures. This ability renders 3c-ALEX ideal for extracting equilibrium and kinetic constants for multi-component interactions (9,10,23).
To determine whether 3c-ALEX can sort and identify molecules independent of FRET, we studied four additional DNA constructs of Fig. 2 A with identical probe stoichiometry but substantially different interprobe distances. The S histograms for all DNA constructs are essentially identical to those of C1 (data not shown); triply labeled species cluster, as in Fig. 3 F. Our results establish that 3c-ALEX can sort species independent of FRET, eliminating the main shortcomings of the existing 3c-FRET methods in studying three-component interactions (9).
3c-ALEX can measure multiple distances using a single solution
Since 3c-ALEX can sort, identify, and select molecules with different probe stoichiometry for further analysis, it can be used to measure up to three distances within triply labeled molecules, and one distance within doubly labeled molecules, even when all species are in the same solution. To explore this possibility, we measured intramolecular distances for the doubly and triply labeled species sorted in Fig. 3 F by selecting each species and examining their E* histograms.
All three doubly labeled species are presented in one-dimensional E* histograms (Fig. 4 A). The distribution for each species is described well by a single Gaussian function, with mean values in agreement with the positions of the probes on the DNA used; is low (0.13 for 19-base-pair (bp) separation), is intermediate (0.46 for 17-bp separation), and is 0 (0.02 for 36-bp separation).
The triply labeled species is presented in a three-dimensional E* histogram that allows simultaneous visualization of three proximity ratios (Fig. 4 B, left); here, only a single species is observed. Mean values of 0.09, 0.44, and 0.10 for , , and , respectively, were determined from three one-dimensional E* histograms (Fig. 4 B, right). These values agree with the positions of the probes on the DNA, as well as with those measured from doubly labeled species (Fig. 4 A). The small differences are due to additional FRET processes in the triply labeled species: decreases by 0.04, due to the G
R FRET, is essentially invariant (since it is independent of the presence of B) and increases due to the BGR FRET. Our results establish that 3c-ALEX can measure multiple distances from multiple species in a single measurement. This feature can be useful in homogeneous assays of ligand-induced conformational changes in a solution containing both a singly labeled ligand (e.g., B-labeled ligand) and a doubly labeled macromolecule (e.g., G-R-labeled macromolecule) (10,11).
3c-ALEX can monitor multiple distance changes within single molecules
To test whether 3c-ALEX can monitor conformational changes within single molecules by observing three intramolecular distances simultaneously, we compared three triply labeled species having different intramolecular distances (C3-a, C3-b, and C3-c (Fig. 5 A)). After sorting and selecting the triply labeled species using the S histograms, we obtained three one-dimensional E* histograms that report on three intramolecular distances (Fig. 5 B). In C3-a (Fig. 5 A, upper), in which the three probes are close to one another, significant FRET occurs between all FRET pairs (proximity ratios , , and of 0.42, 0.27, and 0.63, respectively.)
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20 Å compared to the corresponding distances in C3-a, but did not change the B-R distance. Our results reflect these distance changes: the one-dimensional E* histograms of C3-b (Fig. 5 B, middle) show decreased FRET between B and G and also between G and R (with decreasing from 0.42 to 0.13, and from 0.27 to 0.10), but no change in the FRET between B and R ( remains constant.)
In the same manner, we shifted the R probe by 7 bp toward the 3' end in C3-c (Fig. 5 A, lower), which increased both the B-R and G-R distances by 20 Å compared to the corresponding distances in C3-a, but did not change the B-G distance. Again, our results agree with these distance changes: the one-dimensional E* histograms of C3-c (Fig. 5 B, lower) show that FRET increases between B and R and also between G and R, but not between B and G. Our results verify that 3c-ALEX can monitor three distances (and thus, three distance changes) simultaneously at the single-molecule level, without auxiliary experiments. This capability can help identify the mobile parts of biomachinery and examine the nature, sequence, and kinetics of conformational changes in biomachines (11,30).
Accurate FRET measurements using 3c-ALEX
Recently, we showed that 2c-ALEX measures FRET accurately by achieving full cross-talk and 
(detection-factor (31)) corrections (24). To examine the ability of 3c-ALEX for accurate FRET measurements, we performed cross-talk and detection-factor corrections for five DNA fragments. Cross-talk and detection-correction factors were obtained from singly and doubly labeled species, respectively (Supplementary Material, Theory of 3c-ALEX and -determination). Although each labeled DNA represented a different type of 3c-FRET (Fig. 1 A) and a different set of FRET efficiencies, the measured interprobe distances (Fig. 6, black; detailed and are summarized in Supplementary Material, Table S2) were essentially identical (within 3 Å) and independent of the relative arrangement of the probes. For example, the B-G distance in C2 and C3-b was measured to be 88 Å and 85 Å, respectively, and the B-R distance in C2 and C3-c, 69 Å and 67 Å, respectively.
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). This capability will be useful in using ALEX-based sets of distances to construct models for the structure of large, multi-component complexes (2,28).
Mixture of triply labeled species
In addition to its ability to sort and select molecules based on probe stoichiometry (stoichiometry-based heterogeneity (Fig. 1 B)), 3c-ALEX can sort molecules based on FRET; e.g., it can sort triply labeled species based on their structure (conformational heterogeneity (Fig. 1 B)). To investigate the ability of 3c-ALEX to observe conformational heterogeneity, we studied an equimolar mixture containing triply labeled species (Fig. 7, A–C, C1 and C3-c). After selecting all triply labeled species using the S histogram (Fig. 7 A), we plotted the selected species on E* histogram (Fig. 7 B for a three-dimensional plot and Fig. 7 C for its projections), where two distinct species emerged. The clear separation of these species in the E* histogram allows graphical selection, E* measurement, and molecular counting. The measured proximity ratios for Fig. 7 B agreed well with those measured from solutions containing a single triply labeled species (data not shown), and the ratio of molecules agrees with the initial mixing ratio (575:646 for C1/C3-c).
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–34), and complex processes such as protein and RNA folding (5,6,35,36).
Multi-perspective monitoring of protein translocation on DNA
To demonstrate that 3c-ALEX can monitor multiple conformational changes within large complexes, we monitored the translocation of Escherichia coli RNAP on DNA; this translocation occurs when RNAP breaks its initial interactions with DNA (formed in RNAP-promoter open complex, RPo) and forms an elongation complex (RDe). Previously, we monitored RNAP translocation on DNA by monitoring a single distance within RPo and RDe using the strategies of leading-edge and trailing-edge FRET, both in ensemble (26) and single-molecule experiments (29,37). In leading-edge FRET, forward (i.e., downstream) translocation of RNAP on DNA increases FRET between a donor at the leading edge of RNAP and an acceptor on downstream DNA. In contrast, in trailing-edge FRET, forward translocation of RNAP on DNA decreases FRET between a donor at the trailing edge of RNAP and an acceptor on upstream DNA.
Since 3c-ALEX can report three intramolecular distances simultaneously, we reasoned that we can merge leading- and trailing-edge FRET in a 3c-ALEX assay that monitors simultaneously two protein-DNA and one DNA-DNA distances (Fig. 8 A) in a single RNAP-DNA complex. By incorporating a G probe on RNAP, a B probe on upstream DNA, and an R probe on downstream DNA, RNAP translocation downstream is expected to decrease the BG FRET (equivalent to trailing-edge FRET), and concurrently increase the GR FRET (equivalent to leading-edge FRET); the BR FRET may or may not change. Therefore, we prepared a singly labeled RNAP by attaching a G probe to position 496 of 70 (26,28,38), and a doubly labeled promoter DNA fragment with a B and R probe at its upstream and downstream ends, respectively (Fig. 8 A). Residue 496 on 70 was chosen based on its location on structural models of the open complex (28,39); incorporation of a G probe on residue 496 was expected to yield measurable FRET efficiencies for both trailing- and leading-edge FRET (in a triply labeled open complex), and yield significant and opposite-sign FRET changes upon downstream RNAP translocation. We then formed RPo (Fig. 8 A, upper) and elongation complex RDe,12 (26), where RNAP has translocated downstream by a full turn of DNA relative to its position in RPo (Fig. 8 A), and performed 3c-ALEX.
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70 is not released during the transition of RNAP to elongation (26,29). A substantial release of 70 would have converted one triply labeled molecule (RPo, a B-G-R species) into one singly labeled molecule (free 70, a G-only species) and one doubly labeled molecule (free DNA, a B-R species), changing the S histogram substantially. Moreover, a comparison of E* histograms for triply labeled RPo and RDe,12 showed that for 85% of the complexes, trailing-edge FRET decreases substantially (mean decreases from 0.51 to 0.10; Fig. 8 B, left), whereas leading-edge FRET increases substantially (mean increases from 0.03 to 0.32; Fig. 8 B, middle). These FRET changes correspond to an overall downstream translocation of RNAP on DNA, which increases the distance within the trailing-edge FRET probe pair and decreases the distance within the leading-edge FRET probe pair. This way, the direction of RNAP movement is unequivocally determined with a single measurement. Our results should promote real-time analysis of the conformational changes during transcription and other nucleic-acid processing pathways. |
CONCLUSION AND OUTLOOK |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSION AND OUTLOOKSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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), protein and RNA folding, and multi-step signal-transduction cascades. 3c-ALEX can also aid in ultrasensitive protein-ligand binding assays (40), biosensing (8), and multiplexing (41).
To measure real-time dynamics and reaction trajectories of many individual molecules simultaneously, 3c-ALEX can be extended to immobilized molecules, using either total internal reflection (37,42) or confocal microscopy. Studies of immobilized molecules by 3c-ALEX will directly report on the nature, sequence, coupling, and kinetics of conformational changes occurring during biological mechanisms, providing detailed views of molecular machines at work.
3c-ALEX can also be realized by using three interlaced pulsed-lasers (36,43), which will add fluorescence lifetime information to the current observables and be useful for cross-talk-free multi-color fluorescence imaging microscopy and fluorescence cross-correlation spectroscopy.
Multi-distance analysis by 3c-ALEX is the first step toward n-color-ALEX which, in principle, should enable observation of n-component interactions and determination of up to (n(n – 1)/2) distances within single molecules. The collection of multiple distances (through simple triangulation) will unequivocally determine the probe positions in three-dimensional space, and provide restraints for constructing low-resolution, solution-based structures of large, multi-component complexes intractable due to excessive heterogeneity, limited quantity, or transient nature.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSION AND OUTLOOKSUPPLEMENTARY MATERIALACKNOWLEDGEMENTSREFERENCES |
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This work was funded by a National Research Laboratory grant and a Chemical Genomics grant of the Korean Science and Engineering Foundation and Ministry of Science and Technology to S.K.K., and National Institutes of Health grant GM65382 and GM069709-01A1 and Department of Energy grants FG03-02ER6339 and 04ER63938 to S.W.
Submitted on July 13, 2006; accepted for publication September 11, 2006.
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REFERENCES |
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, excitation of probe B. (A, left) Two-step FRET (
90 Å.
