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Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes
Vanessa C. DeRocco*1, Trevor Anderson*2, Jacob Piehler3, Dorothy A. Erie1, and Keith Weninger2
1Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
2Department of Physics, North Carolina State University, Raleigh, NC, USA
3Universität Osnabrück, Fachbereich Biologie, Osnabrück, Germany

*V.C.D. and T.A. contributed equally to this work.
BioTechniques, Vol. 49, No. 5, November 2010, pp. 807–816
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To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris–nitrilotriacetic acid (Tris-NTA)–conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.

Single-molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), enable measurements of molecular associations and conformational changes during protein-protein and protein–nucleic acid interactions (1). Many important biological processes are amenable to single-molecule studies by fluorescently labeling the molecules of interest and recording fluorescence emissions. Detection of three distinct single-molecule fluorescence signals for multimeric colocalization or multiple FRET couplings have been demonstrated (2-9). Discrimination of single-molecule fluorescence from four distinct dyes is used in a commercial DNA sequencing instrument using zero mode waveguide sample chambers and prism-based spectral dispersion (10,11). This sophisticated instrument has not quantified FRET efficiency at single-molecule level for four dyes. FRET interactions among four dyes on DNA have been recorded with a confocal microscope using photodiodes for single point detection (12). Herein we present a simple modification of the typical two-color total internal reflection microscope(TIRM), which provides detection of single-molecule fluorescence in four distinct spectral channels simultaneously using wide-field imaging, and we demonstrate its capabilities for quantitative FRET studies (Figure 1). Instrument construction is related to that used to characterize polarization and two-color FRET simultaneously (13). We combine this instrument with Tris–nitrilo-triacetic acid (Tris-NTA) dye labeling to introduce a flexible approach for studying dynamics of multimeric complexes.

To dye-label proteins while preserving function, we use the recently developed Tris-NTA-fluorophores (14,15). Ensemble FRET studies (16) used similar mono- and bis-NTA dyes, which bind 6-histidine (His6)–tagged proteins with micromolar affinities. In contrast, Tris-NTA-fluorophores have subnanomolar to nanomolar binding constants for His6 proteins and dissociation rates on the order of 10−3 to 10−4 s−1 (15). The dye-bound state lifetimes of thousands of seconds are sufficient for most single-molecule fluorescence experiments. A fluorescent dye is attached to the Tris-NTA moiety via a six-carbon linker (15) similar to the linker in commonly used covalently linked dyes. This linker allows rotational flexibility for the dye, which is important for quantitative FRET applications. The noncovalent labeling is versatile, easily performed, and enables labeling of proteins that do not tolerate more common labeling approaches. We demonstrate colocalization of a transient binding partner via its Tris-NTA-fluorophore, while simultaneously measuring conformational changes in the substrate using a FRET pair spectrally distinct from the Tris-NTA dye with single-molecule sensitivity for two different biological systems: one monitoring protein-induced DNA bending (yMutSα-mismatched DNA complexes) and the other monitoring protein-induced conformational changes in another protein (SNARE complexes). Both of these systems involve protein-induced conformational changes in the substrate that can be monitored by smFRET; however, independently colocalizing the binding partner confirms assembly of the complex.

Materials and methods


We purchased dye- and biotin-labeled oligonucleotides from Integrated DNA Technologies (Coralville, IA, USA). The 50-bp biotin/TAMRA-labeled oligonucleotide was 5′-biotin-TGTCGGGGCTGGCT-TAAGGTGTGAAATACCTCATCTC-GAGCGTGCCGATA-TAMRA-3′. The Cy5-labeled 19-bp complement was 5′-TATCGGCACCCTCGAGATG-Cy5-3′ (underlining indicates CC base-base mispair). The unlabeled 31-bp complement was 5′-AGGTATTTCACACCTTAAGC-CAGCCCCGACA-3′. The 50-bp and 19-bp oligonucleotides were annealed at ~65°C for 20 min and slowly cooled to 55°C. At 55°C, the 31-bp complement was added, and the mixture was cooled to room temperature. (Figure 2, A and B)

yMsh2-Msh6 protein

Expression and purification of His6-tagged yMsh2-Msh6 (yMutSα) was performed as described (17). Thomas Kunkel (National Institute of Environmental Health Sciences [NIEHS], Research Triangle Park, NC. USA) provided yMsh2 (pAC12 His-Msh2) and yMsh6 (yEpspGal Msh6) plasmids.

SNARE proteins

Expression and purification of full-length syntaxin-1A, soluble syntaxin-1A (1–263), SNAP-25, and soluble synaptobrevin (1–96) were as described (2,18,19). Unless indicated that they were retained, His6 tags were removed with thrombin as verified by SDS-PAGE. Munc-18 was expressed and purified as described (18) without His6 tag removal.

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