Method Article

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

DOI:

10.3791/55623

⸱

May 13th, 2017

 ,  ,  ,  ,  , 
1Department of Chemistry, Clemson University, 2Department of Physics and Astronomy, Clemson University, 3Department of Biochemistry and Molecular Biology, Center for Membrane Biology, Graduate School for Biomedical Sciences, University of Texas Health Science Center

In This Article

Summary

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A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

Abstract

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A protocol on how to perform high-precision interdye distance measurements using Förster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all "dimensions" of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 Å in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 Å for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

Introduction

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A fundamental goal of structural biology studies is to unravel the relationship between the structure and function of biomolecular machines. The first visual impression of biomolecules (e.g., proteins and nucleic acids) occurred in the 1950s through the development X-ray crystallography1,2. X-ray crystallography provides high-resolution, static structural information constrained by the crystal packing. Therefore, the inherent immobility of X-ray structural models shuns the dynamic nature of biomolecules, a factor that impacts most biological functions3,4,5. Nuclear magnetic resonance (NMR)6,7,8 provided an alternative solution to the problem by resolving structural models in aqueous solutions. A great advantage of NMR is its ability to recover the intrinsic dynamic nature of biomolecules and conformational ensembles, which helps to clarify the intrinsic relationships between structure, dynamics, and function3,4,5. Nevertheless, NMR, limited by sample size and large amounts of sample, requires complex labeling strategies for larger systems. Therefore, there is a pressing need to develop alternative methods in structural biology.

Historically, Förster resonance energy transfer (FRET)9 has not taken an important role in structural biology because of the misconception that FRET provides low-accuracy distance measurements. It is the purpose of this protocol to revisit the ability of FRET to determine distances on the nanometer scale, such that these distances can be used for building structural models of biomolecules. The first experimental verification of the R-6 dependence on the FRET efficiency was done by Stryer in 196710 by measuring polyprolines of various lengths as a "spectroscopic ruler." A similar experiment was accomplished at the single-molecule level in 200511. Polyproline molecules turned out to be non-ideal, and thus, double-stranded DNA molecules were later used12. This opened the window for precise distance measurements and the idea of using FRET to identify structural properties of biomolecules.

FRET is optimal when the interdye distance range is from ~0.6-1.3 R0, where R0 is the Förster distance. For typical fluorophores used in single-molecule FRET experiments, R0 is ~50 Å. Typically, FRET offers many advantages over other methods in its ability to resolve and differentiate the structures and dynamics in heterogeneous systems: (i) Due to the ultimate sensitivity of fluorescence, single-molecule FRET experiments13,14,15,16 can resolve heterogeneous ensembles by directly counting and simultaneously characterizing the structures of its individual members. (ii) Complex reaction pathways can be directly deciphered in single-molecule FRET studies because no synchronization of an ensemble is needed. (iii) FRET can access a wide range of temporal domains that span over 10 decades in time, covering a wide variety of biologically relevant dynamics. (iv) FRET experiments can be performed in any solution conditions, in vitro as well as in vivo. The combination of FRET with fluorescence microscopy allows for the study of molecular structures and interactions directly in living cells15,16,17,18,19, even with high precision20. (v) FRET can be applied to systems of nearly any size (e.g., polyproline oligomers21,22,23,24, Hsp9025, HIV reverse transcriptase26, and ribosomes27). (vi) Finally, a network of distances that contains all the dimensionality of biomolecules could be used to derive structural models of static or dynamic molecules18,28,29,30,31,32,33,34,35,36,37.

Therefore, single-molecule FRET spectroscopy can be used to derive distances that are precise enough to be used for distance-restrained structural modeling26. This is possible by taking advantage of multiparameter fluorescence detection (MFD)28,38,39,40,41,42, which utilizes eight dimensions of fluorescence information (i.e., excitation spectrum, fluorescence spectrum, anisotropy, fluorescence lifetime, fluorescence quantum yield, macroscopic time, the fluorescence intensities, and the distance between fluorophores) to accurately and precisely provide distance restraints. Additionally, pulsed interleaved excitation (PIE) is combined with MFD (PIE-MFD)42 to monitor direct excitation acceptor fluorescence and to select single-molecule events arising from samples containing a 1:1 donor-to-acceptor stoichiometry. A typical PIE-MFD setup uses two-pulsed interleaved excitation lasers connected to a confocal microscope body, where photon detection is split into four different channels in different spectral windows and polarization characteristics. More details can be found in Figure 1.

It is important to note that FRET must be combined with computational methods to achieve atomistic-like structural models that are consistent with FRET results26,30. It is not the goal of the present protocol to go over the associated methodology to build structural models with FRET-derived distances. However, these approaches have been applied in combination with other techniques (e.g., small-angle X-ray scattering or electron paramagnetic resonance), giving birth to the field of integrative structural biology43,44,45,46. The current goal is to pave the way for FRET as a quantitative tool in structural biology. As an example, this methodology was used to identify three conformational states in the ligand-binding domain (LBD) of the N-methyl-D-aspartate (NMDA) receptor. The ultimate aim is to overcome the aforementioned limitations and to bring FRET amongst the integrative methods used for the structural determination of biomolecules by providing measured distances with high precision.

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Protocol

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1. PBS Buffer Preparation and Chamber Treatment

NOTE: Wear a laboratory coat and disposable gloves when performing wet chemical experiments. Use eye protection when aligning the laser.

  1. PBS buffer preparation
    1. Dissolve 4.5 g of Na2HPO4, 0.44 g of NaH2PO4, and 3.5 g of NaCl in 400 mL of distilled water. Ensure a pH of 7.5 and sterilize the solution by autoclaving on a liquid cycle for 1 h (depending on the autoclave system).
    2. Take 15 mL of the PBS solution and mix it with 0.1 g of charcoal. Filter the mix by using a regular 20 mL syringe filter with a 0.2 µm pore size. Seal and store the PBS buffer at room temperature.
  2. Microscope chambered cover glass treatment
    1. Add 500 µL of distilled water and 5 µL of polysorbate 20 nonionic surfactant (see the Materials List) to a chambered cover glass system (see the Materials List) and mix well. Let it soak for 30 min. Remove the polysorbate 20 solution and wash the chamber with distilled water twice. Let it dry.
      NOTE: The chamber is now ready to use.

2. DNA Sample Preparation

NOTE: Use designed labeled DNA strands (see the Materials List) for the creation of double-stranded DNA (dsDNA) standard samples. Designed oligos must not have dyes at the end of a polymer in order to avoid artifacts that can compromise the determined distance. The DNA sequence should be chosen to behave as a rigid body.

  1. Add 1.5 µL of a donor labeled DNA strand and 4.5 µL of a complimentary (acceptor-labeled or non-labeled) DNA strand into a microfuge tube and mix them with 24 µL of nuclease-free water.
    NOTE: Depending on the mix of the selected oligonucleotides, the following samples will be generated: no-FRET, low-FRET, or high-FRET dsDNAs.
  2. Hybridize the DNA using a thermal mixer and the following process: 95 °C for 10 min, 90 °C for 10 min, 80 °C for 10 min, 70 °C for 10 min, 60 °C for 10 min, 50 °C for 10 min, 40 °C for 10 min, 30 °C for 10 min, 20 °C for 10 min, 10 °C for 10 min, and holding at 5 °C.
    NOTE: The generated dsDNA standards can be kept in a -20 °C freezer for long-term storage or can be used immediately.

3. Protein Sample Preparation

Note: Starting with recombinant DNA for the expression of the protein of interest in bacterial systems, it is possible to mutate the residues from which the distances are to be measured into cysteines. To do so, use standard site-directed mutagenesis techniques47. To facilitate protein purification, clone the recombinant and mutated DNA into a vector containing a purification tag (e.g., a His-tag). The glutamate subunit 1 ligand-binding domain (LBD) from the NMDA glutamate ionotropic receptor (GluN1) LBD (i.e., NMDA GluN1 LBD cloned into the pET-22b (+) vector) was used.

  1. Protein expression
    1. Transform construct DNA plasmid into the expression system of choice48.
      NOTE: The following steps will assume that the expression of a soluble protein is transformed in Escherichia coli. Purification from, for example, transfected mammalian cells49 or transduced insect cells50, is also possible, and detailed steps can be found elsewhere. Ensure that the E. coli strain selected is appropriate for the protein of interest. For example, the expression of proteins that contain disulfide bridges requires a strain of competent cells with a less-reducing intracellular compartment (see the Materials List).
    2. Inoculate a starter E. coli culture by using a sterile pipette tip to pick up a single transformed colony48. Drop it into 100 mL of selective LB medium (see the Materials List) and allow the culture to grow overnight at 37 °C.
    3. Prepare LB broth (see the Materials List). Autoclave it to sterilize.
    4. Inoculate large-scale cultures of transformed E. coli by adding the overnight culture to 2 L of selective LB medium at a 1:500 ratio.
    5. Over the next few hours, assess the growth of the culture by monitoring the absorbance readings (Abs) of the culture at 600 nm, sometimes referred to as the optical density at 600 nm (OD600), or by using a cell density meter. Note that the readings increase over time.
    6. Induce protein expression with a final concentration of 0.5 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) when the culture reaches an OD600 of 0.7. Shake induced E. coli at 20 °C for 20-24 h.
    7. After protein induction, pellet the E. coli by spinning for 20 min at 3,000 x g and 4 °C. Discard the supernatant and store the E. coli pellet containing the intracellular protein at -80 °C until use.
  2. Protein purification
    1. Lyse E. coli using a lysis method of choice (e.g., sonication, French press, nitrogen cavitation, etc.)51.
    2. Spin down the membrane and cell debris by centrifuging the lysate for 1 h at 185,000 x g and 4 °C.
    3. For a His-tagged protein, load the supernatant onto an equilibrated, nickel-charged, immobilized metal affinity chromatography (IMAC) column using a fast protein liquid chromatography (FPLC) system (see the Materials Table)52.
      NOTE: Equilibration buffer for the NMDA GluN1 LBD: 200 mM NaCl, 20 mM Tris, and 1 mM Glycine, pH 8. Elution buffer for NMDA GluN1 LBD: 200 mM NaCl, 20 mM Tris, 1 mM Glycine, and 400 mM Imidazole, pH 8.
      1. Wash the IMAC column with buffer containing a low amount (~12 mM) of imidazole.
      2. Elute the protein from the IMAC column using a linear gradient of imidazole from 12 mM to 400 mM.
    4. Dialyze the protein overnight in equilibration buffer without imidazole53 by placing the eluate from step 3.2.3.2 into dialysis tubing and submerging it in the equilibration buffer under continuous stirring for 2-3 h. Repeat at least one more time.
      NOTE: Steps 3.2.3-3.2.6 assume the purification of a His-tagged protein. If purifying through some other method, adjust the protocol accordingly.
    5. Quantify the protein amount by taking the absorbance at 280 nm of the dialyzed protein and using Beer's Law (Absorbance unit = ε L c, where ε is the extinction coefficient (M-1cm-1), which can be obtained here54; L is the light path length (cm); and c is the protein concentration (M)).
      NOTE: Various protein quantification assays are available, including the Bradford assay and bicinchoninic acid assay. Both provide accurate results.
  3. Protein labeling
    1. Add donor (maleimide reactive cyan-green dye) and acceptor (maleimide reactive far-red dye) fluorophores to the purified protein at a 1:1:8 protein:donor:acceptor molar ratio.
    2. Incubate the protein and fluorophore mixture on ice for 30 min. Longer incubation times are possible.
    3. Pack a 0.5 mL Ni-Nitrilotriacetic acid (Ni-NTA) agarose column (see the Materials List) and equilibrate it using the same equilibration buffer as in step 3.2.3 while the protein is incubating.
      NOTE: Keep the amount of loaded protein in accordance with the resin binding capacity.
    4. After the 30 min incubation, load the protein/fluorophore mixture onto the column prepared in step 3.3.3 and purify by gravity flow.
    5. Wash off excess fluorophore with 5 mL of equilibration buffer.
    6. Elute the labeled protein by gravity from the column four times with 0.5 mL of elution buffer. Because the protein has already been purified from other proteins, no gradient is necessary.
    7. Check each eluate with a UV-Vis spectrometer to identify which fraction contains the labeled protein. Scan the absorbance from 230-700 nm to be able to ensure that the absorbance peaks from the protein (280 nm) and each fluorophore (493 nm for the cyan-green fluorophore and 651 nm for the far-red fluorophore) are visible in the eluate.
      NOTE: Typically, the protein will elute in fraction 2.
    8. Equilibrate a desalting column (see the Materials Table) with charcoal-treated PBS (step 1.1).
    9. Load the labeled protein onto the desalting column by gravity flow.
      NOTE: Keep the amount of loaded protein in accordance with the selected desalting column capacity55.
    10. Elute using 3.5 mL of charcoal-treated PBS by gravity flow and collect 0.5 mL fractions of the eluate.
    11. Use a UV-Vis spectrometer to scan the absorbance of each eluate from 230-700 nm to identify which fraction contains labeled protein.
      NOTE: Steps 3.3.8-3.3.11 basically serve as a buffer exchange step. Other protocols that serve this purpose are also possible (e.g., extensive dialysis). Alternatively, one could go straight to step 3.3.8 from step 3.3.2.

4. Measurements Needed in Ensemble Conditions (in Cuvette)

  1. Determination of the Förster constant
    1. Scan fD, the fluorophore fluorescence emission (cps), in a fluorimeter by exciting the donor at 15 nm to its maximum absorbance wavelength in order to get the full emission spectrum. Monitor the emission beginning 5 nm after the excitation wavelength and ending 150 nm later. Use magic angle conditions by setting the emission polarizer to 54.7° and the excitation polarizers to 0°56.
      NOTE: For the donor fluorophore used here, the Abs maximum occurs at 490 nm; 475 nm is used as the excitation wavelength, and the emission from 480-650 nm is monitored. For the acceptor, the Abs maximum occurs at 645 nm; 630 nm is used for the excitation wavelength, and the emission from 635-735 nm is monitored.
    2. Use the fluorimeter to perform an excitation scan of the acceptor fluorophore (AbsA) ranging from 400-700 nm and use magic angle conditions by setting the emission polarizer to 54.7° and the excitation polarizers to 0°56. Set the emission monochromator to 15 nm after the maximum-emission wavelength. Normalize to the maximum excitation value.
    3. On published tables, locate the extinction coefficient of the acceptor, εA (M-1 cm-1)56, or use values provided by the manufacturer.
      NOTE: The value published for the acceptor fluorophore used in this manuscript is εA647 = 270,000 cm-1 M-1 56.
    4. Calculate the spectral overlap using J = ΣfD · (AbsA · εA) · λ4, where fD, AbsA, and εA have been defined above λ and is the wavelength (nm). Use a worksheet to list in columns all the wavelength-dependent values obtained in steps 4.1.1-4.1.3. Align them according to wavelength. Perform the summation from the minimum wavelength of the donor emission (λmin) to the maximum wavelength of the acceptor absorbance (λmax).
    5. Calculate the Förster constant (Ro) using the following formula Ro6 = 8.79 x 10-5 · J · κ2 · ΦF,D · n-4, where J is the spectral overlap previously calculated in step 4.1.4, κ2 is the orientation factor, ΦF,D is the fluorescence quantum yield of the donor fluorophore, and n is the refractive index of the medium in which the fluorophore is situated.
      NOTE: Use n = 1.33 (if aqueous buffer is used) and κ2 = 2/3.
    6. Locate the quantum yield value of the donor fluorophore (ΦF,D, environment dependent) on published tables (see Reference 57) and use the value of the spectral overlap obtained in step 4.1.4 to calculate the final value of the Förster constant using the equation from step 4.1.5.
      NOTE: If the quantum yield is not available, follow step 4.2, below, to calculate it. In this case, use ΦF,D = 0.8, which corresponds to a donor lifetime τD,r = 4.0 ns.
  2. Determination of fluorescence quantum yield
    NOTE: The following procedure assumes only dynamic quenching. To consider static quenching, refer to Reference 56. However, PIE-MFD experiments are also useful in determining the quantum yield, even in the case of static quenching (see the Results).
    1. Select a reference fluorophore with similar absorbance and emission profiles for both the acceptor and the donor fluorophores for which the quantum yield (Φr) has been determined.
      NOTE: For the donor, Φr = 0.8 and τr = 4 ns, while for the acceptor, Φr = 0.32 and τr = 1.17 ns, which correspond to the Φr and τr for the cyan-green fluorophore- and the far-red fluorophore-labeled oligonucleotides, respectively57.
    2. Measure the time-resolved fluorescence decay (f(t)) using the time-correlated single-photon counting (TCSPC) method at magic-angle conditions.
    3. Fit the fluorescence decay with a mono- or multi-exponential decay function in the form of f(t) = Σixie-t/τi, where xi is the population fraction and τi is the population fluorescence lifetime.
    4. Calculate the species average lifetimes, 〈τ〉x = Σxiτi, where xi is the population fraction and τi is the population fluorescence lifetime.
    5. Use the formula ΦF,D = 〈τD〉x * Φr / τr to calculate the fluorescence quantum yield of the donor fluorophore by plugging in the fluorescence lifetime and quantum yield of the reference, as well as the fluorescence lifetime of the donor fluorophore.
      NOTE: This method assumes dynamic quenching. For other ΦF,D determinations, follow Lakowicz56.

5. Experiment Alignment for PIE-MFD Single-molecule Detection (SMD)

NOTE: It is better to turn off the lights when taking measurements.

  1. Equipment adjustment (Figure 1)
    NOTE: A home-built MFD setup depicted in Figure 1, with two pulsed lasers and 4 detection channels in an inverted microscope body, is used for this experiment. There are similar commercial systems.
    1. Turn on the 485-nm and 640-nm lasers and all detectors of the MFD setup. Open the software that controls the TCSPC acquisition and lasers. Make sure that the laser repetition rate is 40 MHz.
    2. Set the 485-nm pulsed laser power to 60 µW at an image plane of the 60X 1.2 N.A. water-immersion objective and the 640-nm pulsed laser power to 23 µW in pulsed interleaved excitation mode (PIE-MFD)42.
      NOTE: To set PIE-MFD, the two laser pulses are delayed in the laser controller software. For 485-nm laser excitation, the detection TCSPC channels (TAC channels) are 1-12,499 ("prompt" channel). For 640 nm laser excitation, the detection TCSPC channels (TAC channels) are 12,499-50,000 ("delay" channel).
    3. Add objective immersion liquid (a drop of double-distilled water) between the microscope objective lens and a cover glass slide. To ensure that the image plane is inside the solution and far from the glass surface, turn the adjustment knob one and a half turns after finding the second bright focal point due to the reflection of the lasers at the glass-liquid interface.
    4. Add 1 µL of 100 nM Rhodamine 110 to 50 µL of distilled water to the center of the cover glass. Ensure that the solution is also at the center of the microscope objective.
    5. Adjust the pinhole (size: 70 µm) positions (x and y direction one at a time) while monitoring the photon count rate on the acquisition software to maximize the number of photons detected.
  2. Standard measurement SMD (work in a dark room)
    1. Use the sample from step 5.1.4 and record 120 s of the count rate by clicking the "Start" button on the time-tagged time-resolved (TTTR) control panel in "*.ht3" format58 on the acquisition software.
    2. Compute offline fluorescence correlation spectroscopy (FCS)59,60,61 (i.e., FCS measurement) to determine the characteristic time of diffusion, the number of molecules in the confocal volume, the triplet state kinetics, and the molecular brightness62.
      1. Open the software for FCS (Kristine, MFD suite). Select the experimental settings by clicking "Options" -> "Select Set up." Select a file with similar experimental settings and click "get parameters from file" to read the header information on the file.
      2. Select "Operate" -> "Correlate" to perform FCS.
        NOTE: Make sure that the channel numbers are properly specified and that the "TAC Gate" (TCSPC channels) is checked to select accordingly the prompt or delay channels.
      3. Select "Operate" -> "Global Fit of Correlation Curves" to open the fit routine. Use "equation #24" on the software and click "start."
        NOTE: Equation #24 on the software describes the autocorrelation function (Gc) of freely diffusing fluorescent molecules over a three-dimensional Gaussian illumination profile, such as61:
        Fluorescence correlation spectroscopy equation, G(tc) formula, analytical method.
        where N is the mean number of molecules in the detection volume, xT is the fraction of molecules exerting triplet-state kinetics with the characteristic time tT, tc, is the correlation time, tdiff is the diffusion time related to the geometrical parameter ω, and ω describes the Gaussian illumination profile. After the fit, take note of the diffusion time and the number of molecules in the confocal volume.
         
    3. Add 10 µL of 100 nM Rhodamine 101 into 50 µL of distilled water and mix well. Place this mix on top of the cover glass and ensure that the droplet is at the center of the objective lens. Click the "start" button on the TTTR control panel to record 120 s of data in TTTR format.
    4. Add 1 µL of 100 nM far-red fluorophore into 50 µL of distilled water and mix well. Place this mix at the center of the objective lens. Click the "Start" button and record 120 s of data in TTTR format.
    5. Place 50 µL of distilled water at the center of the objective lens. Click the "Start" button and record 300 s of data in TTTR format.
    6. Place 50 µL of PBS buffer at the center of the objective lens. Click the "Start" button and record 300 s of data in TTTR format.
    7. Take 1 µL of the mix from step 5.1.4 and mix with 50 µL of distilled water. Place this mix on the cover glass. First, click the "Start" button and collect 10 s of data in TTTR mode. Then, analyze the TTTR mode file using the Burst Integration Fluorescence Lifetime (BIFL) analysis software (Paris, MFD suite), as described in step 5.3.
      NOTE: Verify the number of bursts per second from the burst selection and analysis software26,61. If the burst level is around 35 every 10 s, it is appropriate for single-molecule measurement.
    8. Continue recording the count rate in TTTR format for 1.5 h (i.e., TCSPC at SMD) to treat as a single-molecule measurement standard.
      NOTE: Due to the large file size, split raw "*.ht3" files into smaller-size files to load and process using BIFL.
  3. Analysis of standard samples using BIFL
    1. Open the BIFL software (Paris).
    2. Select the setup for PIE in the "confirm set up" automatic pop-up window and read the header by selecting the file with similar experimental settings. Click "get parameters from file." Click "OK." Note that the pop-up window closes and is integrated into the Paris front-end. Click "OK" under "Next."
    3. Choose the files to analyze by clicking "Select" on "Data Path Array" to select the measurement to analyze.
      1. Click on "Green scatter" (for a water measurement), "Green BG" (for a buffer measurement), "Green thick" (for a 2 nM Rhodamine 110 measurement), "Red scatter" (for a water measurement), "Red BG" (for a buffer or water measurement), "Red thick" (for a 20-nM Rhodamine 101 measurement), "Yellow scatter" (for a water measurement), "Yellow BG" (for a buffer measurement), and "Yellow Thick" (for a 2 nM far-red fluorophore measurement).
      2. Click "OK" under "Next."
        NOTE: The "Green" channel corresponds to the signal of the green detectors in the "prompt" TCSPC channels. The "Red" channel corresponds to the signal of the red detectors in the "prompt" TCSCP channels. The "Yellow" channel corresponds to the signal of the red detectors in the delay TCSPC channels.
    4. Click "Adjust" next to "Data cut Burstwise" to adjust single-molecule selection parameters. In the new pop up window, select single-molecule events with two standard deviations from the mean interphoton arrival time ("dt") by changing the interphoton arrival time under "Threshold" and the minimum number of photons per single molecule event under "min. #." Click "Return" to close the pop-up window. Click "OK" under "Next."
      NOTE: The threshold, in ms units, depends on the background count rate. The typical minimum number of photons used is 60.
    5. Adjust the initial fluorescence lifetime "Color fit parameters" (e.g., from, to, and convolution) for the generated fluorescence decay parameters on the "Green", "Red," and "Yellow" colors. In the same window, adjust the "from" and "to" values for "Prompt" and "Delay." Click "Return" to close the pop-up window. Click "OK" under "Next."
      NOTE: Ensure that the 2-color excitation check-box is selected. "From" and "to" correspond to the initial and end bins on the fluorescence decay histogram (TAC channel number). If the initial fit parameters are selected properly, a fit function is added to the fluorescence decays on each channel.
    6. Select the location on the hard drive to which to save all processed ascii files in a parent folder.
      NOTE: Paris processes all selected bursts and creates multiple ascii output files than can be used by other programs for visualization (e.g., Margarita MFD suite).

6. dsDNA Standards and Sample Measurements

  1. Add 500 µL of PBS buffer to a chambered cover glass and place a drop of distilled water between the chamber and the objective lens. Click the "Start" button on the control pedal and collect 5 min of data in TTTR mode to use for analysis.
  2. Take a small amount (usually around 0.1 µL, concentration of around 1 µM) of dsDNA standard, add it to the PBS buffer, and mix well. First, collect 10 s of data by clicking "Start." Then, check the burst to get 35 bursts per 10 s (as in steps 5.2.7 and 5.3). Finally, collect >2 h of data in TTTR format, as described above.
  3. Analyze the collected data for the dsDNA samples, as in step 5.3.3.
  4. Visualize the burst histograms using the MFD suite (Margarita) and display the FRET efficiency versus 〈τD(A)〉f or the FD/FA versus 〈τD(A)〉f.
    1. Open the Margarita software and select "File" -> "Import all *.??4 and *.mti files." Select the parent folder containing various subfolders.
    2. Select the parameters to visualize by clicking next to the "X" (abscissa) of one of the parameters derived from Paris (e.g., tau green or 〈τD(A)〉f); similarly, repeat this for the ordinate "Y" to select the desired parameter to visualize (e.g., FRET efficiency, FD/FA, or SPIE PIE).
      NOTE: In this case, FRET efficiency, FD/FA, or SPIE correct for proper background count rate in the green, red, and yellow channels; for quantum yields of the donor and acceptor; for the detection efficiency ratio (gG/gR); and for crosstalk (α). Here, gG/gR = 3.7 and α = 0.017, depending only on the instrument. Background count rates depend on the buffer used, and quantum yield values are previously determined.
    3. Add a FRET line by opening the "Overlay Equation" window by clicking "Display" -> "Overlay Equation." Select the static FRET line from the pop-up menu. Select the proper donor lifetimes and quantum yield parameters to generate the proper FRET line.
      NOTE: FRET lines for correlating various FRET indicators can be generated.
  5. Determine the correction factor for the acceptor excitation by the donor excitation source (β) using the stoichiometry parameter by displaying FRET efficiency versus stoichiometry (SPIE) in Margarita (Equation 1, below).
    NOTE: β is chosen such that the donor sample has a peak at SPIE = 1.0 in the stoichiometry scale; the acceptor-only sample should have a stoichiometry of SPIE = 0.0, and the dsDNA with both labels should have a stoichiometry of SPIE ~0.5.
    NOTE: Instrument is now ready, and it is possible to measure FRET-labeled samples.
  6. Measure and analyze FRET-labeled samples prepared in section 3 by following steps 6.3-6.4.

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Results

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In typical smFRET experiments using an MFD setup (laser lines: 485 nm at 60 µW and 640 nm at 23 µW, section 5.1), the fluorescence sample is diluted to a low-picomolar concentration (10-12 M = 1 pM) and placed in a confocal microscope, where a sub-nanosecond laser pulse excites labeled molecules freely diffusing through an excitation volume. A typical confocal volume is <4 femtoliters (fL). At such low concentrations, only single molecules are detected one at a time. The emi...

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Discussion

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In this work, the protocol to align, calibrate, and measure interdye distances with high precision using PIE-MFD single-molecule FRET experiments is presented. By carefully calibrating all instrumental parameters, one can increase the accuracy of the measured distances and reach Angstrom accuracy. To do so, various multidimensional histograms are used to analyze and identify populations for further characterization. Using the mean macro time to verify the stability of the measured samples, it is possible to correct for d...

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Disclosures

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All the authors declare that they have no competing financial interests with the contents of this article.

Acknowledgements

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VJ and HS acknowledge support from NIH R01 GM094246 to VJ. HS acknowledges start-up funds from the Clemson University Creative Inquiry Program and the Center for Optical Materials Science and Engineering Technologies at Clemson University. This project was also supported by a training fellowship from the Keck Center for Interdisciplinary Bioscience Training of the Gulf Coast Consortia (NIGMS Grant No. 1 T32GM089657-05) and the Schissler Foundation Fellowship for Translational Studies of Common Human Diseases to DD. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
charcoalMerck KGaAK42964486 320
syringe filterFisherbrand09-719Csize: 0.20 µm
chambered coverglassFisher Scientific1554091.5 borosilicate glass, 8 wells
microscope cover glassFisher Scientific063014-9size: 24 x 60-1.5
Nuclease free waterFisher Scientific148859nuclease free
tween-20Thermo Scientific2832010% solution of Polysorbate 20
acceptor DNA strand (High FRET)Integrated DNA Technologies1781248955´-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG AT(Cy5)C GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)
acceptor DNA strand (Low FRET)Integrated DNA Technologies1779564245´-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TT(Cy5)A AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)
donor DNA strandIntegrated DNA Technologies1779514375´ -d(GCA ATA CTT GGA CTA GTC TAG GCG AAC GTT TAA GGC GAT CTC TGT TT(Alexa488)A CAA CTC CGA AAT AGG CCG)
DNA strand (No FRET)Integrated DNA Technologies5´ -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)
thermal cyclerEppendorfE6331000025nexus gradient
Alexa Fluor 488 C5 MaleimideThermo ScientificA10254termed cyan-green fluorophore in the manuscript
Alexa Fluor 647 C2 MaleimideThermo ScientificA20347termed far-red fluorophore in the manuscript
Rhodamine 110Sigma-Aldrich83695-250MG
Rhodamine 101Sigma-Aldrich83694-500MG
LB Broth, MillerFisher ScientificBP1426For culture of E. coli
AmpicillinSigma-AldrichA0166Used at 100 µg/mL final concentration in selective LB medium to maintain plasmid selection
Tetracyline Calbiochem58346Used at 12.5 µg/mL final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation
KanamycinFisher ScientificBP906-5Used at 15 µg/mL final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation
Origami B(DE3) Competent CellsMillipore70837-3Competent E. coli cells for expression of protein with disulfide bridges
Isopropyl-β-D-thiogalactopyranoside (IPTG)Fisher ScientificBP1755For induction of E. coli protein expression
HiTrap Chelating HPGE Life Sciences17-0409-01For Large-scale FPLC Purification of His-tagged protein
ImidazoleSigma-Aldrich56749
Ni-NTA Agarose Qiagen30210
PD-10 Desalting ColumnGE Life Sciences17-0851-01
AktaPurifierGE Life Sciences28406264FPLC Instrument
Dialysis tubingSpectrum labs13256215 kD MWCO 24 mm Flath width, 10 meters/roll
DichroicsSemrockFF500/646-Di01-25x36500/646 BrightLight
50/50 Beam splitter polarizerQioptiq Linos G33 5743 00010 x 10 film polarizer
Green pass filerChromaET525/50mET525/50m 25 mm diameter mount
Red pass filterChromaET720/150mET720/150m 25 mm diameter mount
Power MeterThorLabdPM200
UV-Vis spectrophotometerVarianCary300Bio
Fluorolog 3 fluorometerHoribaFL3-22-R3
Fluorohub TCSPC controllerHoribaFluorohub-BTCSPC electronics for ensemble measurements
NanoLed 485LHoriba485LBlue diode laser
NanoLed 635LHoriba635LRed diode laser
Olympus IX73 MicroscopeOlympusIX73P2FMicroscope frame
PMA 40 Hybrid DetectorPicoQuant GmbH932200, PMA 40Optimized for green detection
PMA 50 Hybrid DetectorPicoQuant GmbH932201, PMA 50Optimized for ed shifter sensitivity
485 nm laserPicoQuant GmbHLDH-D-C-485
640 nm laserPicoQuant GmbHLDH-D-C-640
Hydraharp 400 and TTTR acqusition softwarePicoQuant930021Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software
SEPIA II SLM 828 and SEPIA softwarePicoQuant910028Laser driver for picosecond pulses , includes SEPIA software controller.
computerDelloptiplex 7010cpu: i7-3770 ram:16GB
FRET Positioning and Screening (FPS) softwareHeinrich Heine UnviersityIt include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html
MFD suiteHeinrich Heine UnviersityIt includes the BIFL software package Paris; Margarita for visualization of the multiparameter hisotrams, and Probability Distribution Analysis software availabel at http://www.mpc.hhu.de/software/software-package.html

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$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
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FRETSingle molecule FRETMulti parameter Fluorescence DetectionInterdye Distance MeasurementBiomolecule Structure DeterminationNMDA ReceptorLigand binding DomainConformational StatesFluorescence Resonance Energy TransferPrecision Distance Measurements

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