The sequential and simultaneous FRET and colocalization experiments were performed with a prism-type TIRM, a Quadview splitter containing three dichroic mirrors, and an emCCD, allowing the simultaneous excitation and detection of the fluorescence from 4 dyes with emission spectra characteristic of Cy2, Cy3, Cy5 and Cy7 ( and methods). We first demonstrated this instrument by examining DNA bending induced by yMutSα. MutS homologs are responsible for recognizing and signaling repair of base-base mismatches and insertion-deletions in newly replicated DNA (25
). MutS-mismatch complexes adopt multiple conformations with different degrees of DNA bending (from unbent to bend angles of ~ 90°) (20
). It has been suggested that each bending state may have a different repair signaling potential (20
Biotinylated DNA (50 base pairs) labeled with a FRET donor (TAMRA) and acceptor (Cy5) separated by 19 base pairs with a CC mismatch approximately halfway between the two fluorophores was tethered to a streptavidin-coated quartz surface (). Because yMutSα contains over 30 cysteines, site-specific labeling using maleimide dye methodology is not feasible. Consequently, 6His-tagged-yMutSα (tagged on the N-terminal of Msh2) was non-covalently labeled using tris-NTA-OG (OG-yMutSα) (14
). OG-yMutSα complexes were prepared before addition to CC-mismatch-DNA coated surfaces (methods). DNA bending was monitored by continuously exciting TAMRA with 532 nm illumination and measuring FRET to Cy5. The presence of OG-yMutSα was monitored by measuring OG emission under either continuous (data not shown) or pulsed () 473 nm illumination. Despite using an oxygen scavenging system, a shutter-pulsed scheme was required to allow longer observation intervals before OG photobleached.
After leakage subtraction and gamma correction (Methods), free CC-mismatch DNA molecules exhibit constant FRET efficiencies (), with an average FRET ~ 0.35 (). Unlabeled yMutSα binding increases FRET between TAMRA and Cy5, but yields no blue emission (); whereas, binding of OG-yMutSα increases both FRET and the blue emission, consistent with the presence of the NTA-OG moiety (). In addition, using a shuttered excitation (), the presence of OG-yMutSα can be observed for long times indicating that the OG-NTA moiety remains stably bound to MutSα during the time course of the experiment. These long bound state lifetimes for tris-NTA dyes are consistent with ensemble experiments using NTA-dyes (14
). In the absence of MutSα, the histogram of CC-DNA FRET efficiency vs
. OG-yMutSα emission intensity for many molecules () shows FRET efficiencies around 0.35 and OG emission near zero. In the presence of OG-MutSα, one FRET emission peak overlaps with free DNA and another population shifted to higher FRET values (~0.6 gamma corrected) indicating yMutSα-induced DNA bending. A significant population of DNA without bound protein is expected because yMutSα is present at 5 nM, well below the 54 nM KD
(determined by fluorescence anisotropy). The higher FRET population can be divided into those with low or high OG emission. Those higher FRET events with low blue intensities suggest that unlabeled yMutSα is bound, and those with higher blue (15% of molecules) intensities confirm bound OG-yMutSα. To assess if tris-NTA-dye-labeling affects DNA bending, we compared DNA bending distributions for labeled and unlabeled MutSα and found no significant differences (). These data demonstrate that fluorescently tagged yMutSα is active.
To demonstrate the method’s flexibility, we examined interactions among neuronal SNARE proteins, which mediate membrane fusion (28
). For neuronal SNAREs, two membrane proteins (syntaxin and synaptobrevin) on distinct cellular compartments assemble with a third SNARE protein (SNAP-25) to form a heteromeric complex of 4 parallel α-helices called the SNARE complex, which crosslinks the fusing membranes through the transmembrane domains of the proteins ().
Noncovalent labeling allows dyes to be added to samples in situ, as we demonstrated for membrane-incorporated SNARE complexes. 30 nM Ni3-tris-NTA-OG was introduced above a phosphatidylcholine bilayer that contained SNARE complexes tethered by syntaxin’s transmembrane domain (). The complexes were tagged before assembly with Alexa647-maleimide covalently linked to S61C of synaptobrevin. We used simultaneous 473 nm and 635 nm illumination to identify individual Alexa647 labeled complexes by red emission while NTA-OG binding was assessed by blue emission (). NTA-OG colocalized with over 25% of SNARE complexes when the 6His was present on synaptobrevin, and was reduced to background levels (1–4%) in controls ().
Alternately, covalent labeling with 4 dyes can be used to structurally characterize this multiprotein complex. We prepared SNARE complexes with one donor-acceptor pair (Alexa 488/Alexa 555) at the N-terminal end of the α-helical bundle and a distinct donor-acceptor pair (Alexa 647/Cy7) at the C-terminal end. The SNARE complex is a 2 nm x 10 nm rod-like structure, so high FRET is expected from each pair, but little coupling is expected between the two pairs. Under continuous illumination with both blue and red lasers we simultaneously observed two independent FRET pairs on individual complexes ().
The SNARE complex is also a binding platform for other proteins that regulate membrane fusion. We investigated Munc-18 binding to both syntaxin and to the SNARE complex (28
). In the first SNARE experiment (), syntaxin was covalently labeled with dyes at cysteine mutations in the C-terminal end of the SNARE domain and the N-terminal end of the 3-helix bundle that were designed to yield low (but non-zero) FRET (30
) when the two domains connected by a flexible linker are unbound (open syntaxin) and high FRET when they bind (closed syntaxin). Syntaxin was reconstituted into supported lipid bilayers and Alexa488 labeled Munc-18 was added in solution above the bilayer. Using sequential red-blue-green laser illumination (top axis, ) we observed complexes where blue emission indicated the presence of Munc-18 and FRET in the green and red channels reported the conformational state of syntaxin. We confirmed that Munc18 binds to syntaxin in the closed conformation, as expected (30
We also observed Munc-18 interacting with the full SNARE complex (29
). Soluble SNARE complex was co-encapsulated with Alexa488-Munc-18 in surface-tethered, 100 nm liposomes (). The structure of this 4-protein complex is as yet undetermined. For this experiment, the SNARE complex was labeled with a donor dye (Alexa 555) in the 3-helix bundle of syntaxin and with an acceptor dye on synaptobrevin in the central region of the SNARE bundle. We screened liposomes for the presence of Alexa488-Munc-18 through blue dye emission under pulsed illumination and then used FRET in only those liposomes to examine the spacing between syntaxin’s 3-helix bundle and the core SNARE complex (). Measuring FRET from additional label attachment locations will allow us to put constraints on the overall conformation of the Munc18:SNARE complex.
Our development of a 4-color single molecule fluorescence instrument for measurements on immobilized biomolecules with wide-field imaging will allow single molecule studies of increasingly complex systems. The ability to colocalize binding partners at complexes while also monitoring conformational changes in other parts of the complex via FRET or to simultaneously monitor two FRET pairs on a single complex will enable studies of more complicated molecular assemblies than current approaches. This ability to directly correlate transient accessory binding to dynamic conformational transitions provides a new avenue for studies of biological signaling pathways. Simultaneous quantification of fluorescence emission from 4 dyes interacting through FRET has the potential to report 6 distinct distances on a single molecular complex.
Furthermore, the demonstration that tris-NTA dye conjugates can label 6His-tagged proteins for single molecule studies significantly expands the capability for labeling proteins. Non-covalent labeling at a terminally located affinity tag can minimize the possibility of destroying function in proteins that are susceptible to structural perturbation by point mutations for dye attachment. The tris-NTA labeling approach also avoids the long incubation times required for most covalent labeling strategies and allows in situ labeling. The bound state lifetimes of the tris-NTA dyes to the 6His-tagged proteins are sufficiently long to permit monitoring of many protein-protein and protein-DNA interactions.
The ease of modifying an existing two-color TIRM by using a commercially available 4 color splitting device and its affordability as well as the flexibility of non-covalent dye labeling suggest this method could be adopted by many research groups.