Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.
Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule Förster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.
Single-molecule methods based on fluorescence detection provide high signal-to-noise ratios1. FRET is a spectroscopic technique that can measure distances in the range of 1–10 nm, rendering this technique as a molecular ruler for measuring distances in the nanometer range2,3. The absorption spectrum of the acceptor has a partial spectral overlap with the donor’s emission spectrum at the shorter wavelength end. FRET is mediated by the radiation-less energy transfer between a donor and acceptor pair, whereas the efficiency of energy transfer is dependent on the distance and orientation of the acceptor4.
Several approaches have been implemented to minimize the background and improve the detection efficiency of the fluorescence signal5,6. One approach is confocal microscopy, in which a pinhole restricts the excitation spot to a size below the diffraction limit7. Another approach is total internal reflection fluorescence (TIRF), which is a wide-field illumination technique in which the light is directed off-axis above a critical angle8. The light is then totally internally reflected at the interface between the glass and aqueous solution, generating an evanescent wave that only illuminates the fluorophores attached to the glass surface and prevents background from the fluorophores in the rest of the solution.
In confocal microscopy, the molecules can be either freely diffusing or surface immobilized. The attained temporal resolution can be within microseconds to several milliseconds9. The confocal detection for a single molecule is performed by single-photon avalanche diode (SPAD) and point-by-point scanning of the region of interest10. In TIRF, a time-series of a few hundreds of molecules immobilized on the surface is recorded by a position-sensitive two-dimensional charge coupled detector (CCD). The CCD amplifies the fluorescence signal either by intensified phosphor screen and microchannel plate or on-chip multiplication of photoelectrons (EMCCD). The temporal resolution is dependent on the readout speed and quantum efficiency of the CCD and usually on the order of few tens of milliseconds6.
HJ is a central intermediate in DNA repair and recombination11,12,13,14. HJ has two continuous and two crossing strands that connect between the continuous strands without intersecting each other. HJ exists in solution as X-stacked conformers, which undergo continuous isomerization by the continuous strands becoming crossing and the crossing strands becoming continuous in the other conformer15. Isomer preference of the HJ is dependent on the core sequence and ionic environment and has been extensively studied by FRET16,17,18,19.
GEN120 is a monomeric protein in solution21 and requires dimerization to cleave the HJ, thus allowing proper separation of the recombined strands22,23. The stacking conformer preference of the HJ influences the outcome of genetic recombination by setting the orientation of the resolution by the HJ resolvases24. Understanding how GEN1 binds the HJ, coordinates the two incisions, and ensures its full resolution have all been under intensive study21,22,23,25,26,27,28,29,30.
In this study, an objective based TIRF set-up is used as described previously31. Two-color alternating excitation (ALEX) is applied to determine the conformational changes upon the interaction of GEN1 with fluorophore labeled HJ. ALEX produces 2D histograms based on two ratiometric parameters FRET efficiency E, which is donor-acceptor distance-dependent, and the stoichiometry parameter S, which measures the donor-acceptor stoichiometry32. ALEX enables the sorting of fluorescent species based on the stoichiometries of the fluorophores including donor-only, acceptor-only, and mixed subpopulations. ALEX can extend the use of FRET to the full range and can detect differences in fluorophore brightness and oligomerization as well as monitor macromolecule-ligand interactions33.
It is found that GEN1 consistently succeeds in resolving the HJ within the lifetime of the GEN1-HJ complex. The time-dependent conformational changes are derived from the time-traces of individual molecules, while the histograms represent the distribution of the underlying populations. Using time-lapse single-color FRET, fast on-rates and slow off-rates for the GEN1 dimer are demonstrated, which increase the affinity of the assembled GEN1 dimer at the first incision product.
1. Preparation of surface-functionalized coverslips
2. Preparation of flow cell
3. Preparation of oxygen scavenging system (OSS)
4. Preparation of fluorescently labeled HJs
5. Protein expression and purification of GEN1
6. Single-molecule FRET experiments
NOTE: The smFRET experiments are performed on a custom-built objective based TIRF set-up (Figure 1C) described previously31.
7. Electrophoretic mobility shift assays (EMSA)
Conformer bias and isomerization of the HJ
The isomerization of HJ has been extensively investigated by FRET through the labeling of two adjacent arms of the junction17,18,39. The donor (Cy3) and acceptor (Alexa Fluor 647) are positioned at the two neighboring arms, R (strand 2) and X (strand 3), respectively (Figure 2A). The stacked-X isomers were assigned by their two continuous strands [i.e., Iso(1,3) or Iso(2,4)]. The ALEX FRET histogram of adjacent-label X0 shows two peaks that correspond to interchanging of the more abundant Iso(1,3) (E ~0.75) and less abundant Iso(2,4) (E ~0.40) (Figure 2B).
Single-color FRET is used to acquire time-traces for recording the rapid conformational changes in the free HJ with high temporal resolution ~10 ms via reducing the used area of the EMCCD2 camera. A representative single-color FRET time-trace of X0 junction shows the transitions between high and low FRET isomers (Figure 2B). The isomerization rates kIso(1,3)-Iso(2,4) and kIso(2,4)-Iso(1,3) obtained from the dwell time histograms of Iso(1,3) and Iso(2,4) (Figure 2C) are consistent with those reported previously17.
SMFRET demonstrates active distortion of the HJ by GEN1
HJ undergoes structural rearrangement upon binding to GEN122. Thus, the spacing between the donor and acceptor is similar in both Iso(1,3) and Iso(2,4) (Figure 3A). The smFRET binding assays were carried out in the presence of Ca2+ to prevent cleavage of the HJ. FRET histograms of the adjacent-label X0 junction at different GEN1 concentrations were acquired by ALEX (Figure 3B). The histogram is fit to two Gaussian functions: one corresponding to the free high FRET Iso(1,3), and the other corresponding to the bound GEN1-HJ population after subtracting the contribution of the Iso(2,4) from the low FRET peak.
At saturating GEN1 concentration, the FRET histogram of X0 has only a single low FRET peak corresponding to GEN1 bound to either isomer of the HJ as predicted by the model22. The apparent monomer dissociation constant (Kd-monomer-app) is determined from the hyperbolic fit of the percentages of GEN1-bound population as a function of GEN1 concentration (Figure 3C). The adjacent-label nk-X0 represents a singly nicked version HJ that mimics the product after the first incision reaction. Due to the relief of stacking strain by the simulated nick, nk-X0 is a non-isomerizing structure40 as evident from the single substrate peak at E ~0.40, unlike X0 (Figure 3D vs. Figure 3B). The structure of GEN1-nk-X0 complex is similar to that of the GEN1-X0 complex, as indicated by the similarity in FRET efficiencies (E ~0.25 for nk-X0 and 0.32 for X0) (Figure 3D vs. Figure 3B). The strong binding of GEN1 monomer to nk-X0 is demonstrated by the 40-fold lower Kd-monomer-app value than that of X0 (Figure 3E vs. Figure 3C). This tight binding may act as a safeguard mechanism against the incomplete resolution of the HJ in the unlikely event of the dissociation of GEN1 dimer or one of its monomers.
Stepwise binding of GEN1 monomer to the HJ
The binding of GEN1 monomer to the HJ followed by dimer formation is a unique feature for the eukaryotic HJ resolvase GEN1 compared to prokaryotic resolvases, which exist in dimeric form in solution21,23,41. EMSA of GEN1 at 50 pM X0 shows the stepwise association of GEN1 into higher order complexes, as indicated by the roman numerals in the upper panel (Figure 4A). The dissociation constant of GEN1 monomer determined by EMSA (Kd-monomer-EMSA) coincides with the dissociation constant from the smFRET binding assay Kd-monomer-app (Figure 4A and Figure 3C, respectively). The quantification of band II is used to calculate the equilibrium dissociation constant of GEN1 dimer (Kd-dimer-EMSA). EMSA of GEN1 at 50 pM nk-X0 demonstrates the prominent monomer binding as indicated by the very low Kd-monomer-app-EMSA which is 30-fold lower than that of X0, while its Kd-dimer-EMSA is comparable to that of X0 (Figure 4B).
Further evidence that GEN1 monomer binds and distorts the HJ is the observation of a significant number of traces of uncleaved particles with stable low FRET state (Figure 4C) in the presence of Mg2+ at low GEN1 concentrations. The number of these traces decreased upon increasing GEN1 concentration. The resolution of the HJ is driven by the tight binding of GEN1 monomer, which supports dimer formation. The monomer binding is observed in the time-traces of the uncleaved nk-X0 in Mg2+, which extends until few nanomolar concentration (Figure 4D). The GEN1 monomer binds tightly to safeguard nk-X0, eventually ensuring full resolution through dimer formation.
SMFRET resolution assay of the HJ
The term “cleavage” in smFRET assays is used interchangeably with “resolution” of the HJ, since in this assay only the product release that follows the second cleavage event is detected. The events are recorded by time-lapse single-color excitation to minimize photobleaching of the photo-sensitive acceptor over the acquisition time of ~1.3 min.
The schematic in Figure 5A illustrates the incisions of strands 1 and 3 of X0 Iso(1,3) after the binding and distortion by GEN1 of an X0 attached to the functionalized glass. Both donor and acceptor go into solution resulting in the loss of their signals after the HJ resolution. The first and second incisions are decoupled in nk-X0, which exemplifies a prototype for the partially resolved HJ. Upon binding of GEN1, nk-X0 adopts a similar structure to X0. The resolution proceeds by a single incision in strand 1, as illustrated in Figure 5B.
The simultaneous departure of the donor and acceptor after a stable low FRET state in traces of resolved X0 occurred without the emergence of an intermediate FRET (E = ~0.40) indicates that complete resolution occurs within the lifetime of the GEN1-HJ complex (Figure 5C). Therefore, these results suggest that the HJ resolution occurs within the GEN1-HJ complex lifetime. The resolution of nk-X0 also proceeds after structural rearrangement and concludes by the departure of the duplex carrying two fluorophores (Figure 5D) similar to X0.
Kinetics of GEN1 dimerization on GEN1 monomer bound HJ
Time-lapse smFRET measures τbefore-cleavage which mainly includes the time required for dimer formation and resolution of the HJ after the distortion by GEN1 monomer. Applying this technique, direct evidence is provided to support the claim that dimer formation is required for the resolution of both X0 and nk-X0, since the distribution of τbefore-cleavage is GEN1 concentration-dependent.
The apparent rate of the HJ resolution (kapp) is defined as the inverse of the mean of τbefore-cleavage at the respective GEN1 concentration. The term “apparent” is used to describe the rate of the HJ resolution, since the possibility that GEN1 remains bound to the product after the HJ resolution cannot be excluded.
The probability density functions (PDF) of the τbefore-cleavage distributions of X0 (Figure 6A) reflect the time for dimer formation, which is longer at low GEN1 concentrations, then shorter at higher GEN1 concentrations. The association and dissociation rates for the dimer, kon-dimer and koff-dimer, respectively, are determined from a bi-exponential model30. Also, the PDFs of nk-X0 (Figure 6B) show a similar distribution to X0 indicating the requirement for dimer formation.
The plot of kapp versus GEN1 concentration was fitted to a hyperbolic function. The apparent catalysis rate constants (kMax-app) of X0 and nk-X0 are 0.107 ± 0.011 s-1 and 0.231 ± 0.036 s-1, respectively (Figure 6C). The plots of kapp for X0 and nk-X0 junctions intersect at GEN1 concentration ~5.6 nM because of the faster kMax-app and slower kon-dimer of the nicked compared to the intact junction.
In summary, the relatively fast kon-dimer and slow koff-dimer lead to the progression of the forward reaction towards HJ resolution once the dimer is formed. The strong binding of GEN1 monomer to the nk-X0 junction constitutes a fail-safe mechanism against any unlikely aborted second cleavage or helps to pick up any incompletely unresolved HJs left behind by primary resolution pathways in the cell.
Figure 1: Single and multiple-channel flow cells and layout of the optical set-up.
(A) Schematic of the single-channel flow cell. (B) Schematic of the six-channel flow cell. (C) Layout of the optical set-up depicting the excitation sources, TIRF objective, dichroic mirror installed inside the filter cube, and emission filters used in the image splitter device. Please click here to view a larger version of this figure.
Figure 2: Conformer bias and isomerization of the HJ observed by FRET.
(A) Isomerization of the adjacent-label X-stacked HJ conformers named after the two continuous strands. The strands are numbered, while the arms are denoted by letters. The incision sites are shown by arrows. The positions of the donor (green) and acceptor (red) and the change in FRET upon isomerization are indicated. (B) Right panel: FRET time-trace (black) and idealized FRET trace (red) of X0 at 50 mM Mg2+. Left panel: FRET histogram of X0 at 50 mM Mg2+. The fluorescence intensities of the donor (green) and acceptor (red) are shown below. (C) The dwell time histograms of adjacent-label X0 Iso(1,3) and Iso(2,4) were fitted to single-exponential functions to determine the isomerization rates. The uncertainties indicate the 95% confidence interval of the fit. This figure has been modified from previously published literature30.
Figure 3: Active distortion of the HJ by GEN1.
(A) Structural modification of adjacentlabel HJ based on the proposed model22. (B) ALEX FRET histogram of adjacent-label X0 has a major high FRET peak (E = ~0.6) corresponding to Iso(1,3) and lower FRET peak (E = ~0.4) for Iso(2,4). The entire histogram is fit to two Gaussian functions: one corresponding to the free high FRET Iso(1,3), and the other corresponding to the bound population minus the initial contribution of Iso(2,4) to the total population. (C) The apparent monomer dissociation constant (Kd-monomer-app) is determined from a hyperbolic fit of the percentages of GEN1-bound populations as a function of GEN1 concentration. (D) FRET histograms of the adjacent-label nk-X0 at different GEN1 concentrations. The area under the low FRET (E = ~0.25) Gaussian corresponds to the percentage of the bound population. (E) The Kd-monomer-app of nk-X0 is determined from the hyperbolic fit of GEN1-bound population. The error bars represent the standard deviations from two or more experiments. This figure has been modified from previously published literature30. Please click here to view a larger version of this figure.
Figure 4: Stepwise binding of GEN1 to the HJ.
(A) Electrophoretic mobility shift assay (EMSA) of GEN1 at 50 pM X0. Upper panel: the roman numerals indicate the number of GEN1 monomers in the complex. Lower panel: binding of GEN1 monomer to X0. The apparent dissociation constants were obtained from a sigmoidal fit of the respective species and represent the average of two experiments. (B) EMSA of GEN1 at 50 pM nk-X0 demonstrates the prominent monomer binding as indicated by the very low Kd-monomer-app-EMSA. (C) FRET time-trace of bound but uncleaved adjacent-label X0 in Mg2+. Donor excitation for ~1.3 min was performed, followed by direct acceptor excitation (shaded pink region). (D) FRET time-trace of bound but uncleaved adjacent-label nk-X0 in Mg2+. This figure has been modified from previously published literature30. Please click here to view a larger version of this figure.
Figure 5: SMFRET resolution assay of the HJ.
(A) Schematic of the adjacent-label X0 Iso(1,3) after distortion by GEN1. The substrate is attached to the functionalized surface via biotin/avidin linkage. The dissociation of GEN1 after the two incisions results in the loss of both donor and acceptor that go into solution. (B) Schematic of the resolution of adjacent-label nk-X0 by cleaving strand 1. (C) Time-trace (black) at 2 mM Mg2+ of the cleavage of Iso(1,3). The onset of GEN1 binding forms a stable low FRET state until the FRET signal is abruptly lost due to cleavage. Correspondingly, the increase in the donor and the decrease of acceptor fluorescence intensities upon GEN1 binding is followed by the simultaneous disappearance of the fluorescence from both dyes upon cleavage. (D) Similarly, the time-trace of nk-X0 shows a stable low FRET state upon GEN1 binding which is concluded by the abrupt loss of the FRET signal. This figure has been modified from previously published literature30. Please click here to view a larger version of this figure.
Figure 6: Kinetics of GEN1 dimerization on GEN1 monomer bound HJ.
(A) The probability density function (PDF) plot of the τbefore-cleavage distribution of X0 illustrates its dependence on GEN1 concentration. Dwell times of the low FRET state (τbefore-cleavage) at the respective GEN1 concentration were obtained from two or more experiments and used to obtain average rates (kapp). The listed kapp rates are determined from the inverse of the mean τbefore-cleavage at the respective GEN1 concentration. The association (kon-dimer) and dissociation (koff-dimer) rates for dimer formation are calculated from a bi-exponential model38. The errors represent SEM of kapp. (B) The PDF plot of the τbefore-cleavage distributions of nk-X0 and the respective kapp rates. (C) Plot of kapp versus GEN1 concentration fitted to a hyperbolic function to determine the apparent catalytic rate (kMax-app). The plot of kapp for X0 and nk-X0 illustrates the faster initial kapp of X0 which is then surpassed by nk-X0 above [GEN1] ~5.6 nM. This figure has been modified from previously published literature30. Please click here to view a larger version of this figure.
Buffer | Compostion | ||
Binding buffer | 40 mM Tris-HCl pH 7.5, 40 mM NaCl, 2 mM CaCl2, 1 mM DTT, 0.1% BSA and 5% (v/v) glycerol | ||
Buffer A | 20 mM Tris-HCl pH 8.0, 1 mM DTT and 300 mM NaCl | ||
Buffer B | 20 mM Tris-HCl pH 8.0, 1 mM DTT and 100 mM NaCl | ||
Buffer C | 20 mM Tris-HCl pH 8.0 and 1 mM DTT | ||
Cleavage buffer | 40 mM Tris-HCl pH 7.5, 40 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.1% BSA and 5% (v/v) glycerol | ||
EMSA binding buffer | 40 mM Tris-HCl pH 7.5, 40 mM NaCl, 1 mM DTT, 2 mM CaCl2, 0.1 mg/ml BSA, 5% (v/v) glycerol and 5 ng/µl Poly-dI-dC | ||
Imaging buffer (binding) | 40 µL (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (4 µM), 60 µL PCA (6 nM), 60 µL PCD (60 nM) and 840 µL of Binding buffer | ||
Imaging buffer (cleavage) | 40 µL (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (4 µM), 60 µL PCA (6 nM), 60 µL PCD (60 nM) and 840 µL of Cleavage buffer | ||
Lysis buffer | 20 mM Tris-HCl pH 8.0, 10 mM β-mercaptoethanol, 300 mM NaCl and 2 mM PMSF | ||
PCD storage buffer | 100 mM Tris-HCl pH 7.5, 1 mM EDTA, 50 mM KCl and 50% glycerol | ||
storage buffer | 20 mM Tris-HCl pH 8.0, 1 mM DTT, 0.1 mM EDTA, 100 mM NaCl and 10% glycerol | ||
TBE buffer | 89 mM Tris-HCl, 89 mM Boric acid and 2 mM EDTA | ||
TE100 buffer | 10 mM Tris.HCl pH 8.0 and 100 mM NaCl | ||
Tris-EDTA buffer | 50 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0 |
Table 1: The list of buffers and their compositions used in this study.
Oligo | Sequence | ||
X0-st1 | ACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCCCACCTGCAGGTTCACCC | ||
X0-st2 | GGGTGAACCTGCAGGTGGG/iCy3/AAAGATGTCCATCTGTTGTAATCGTCAAGCTTTATGCCGT | ||
X0-st3 | ACGGCATAAAGCTTGACGA/iAF647-dT/TACAACAGATCATGGAGCTGTCTAGAGGATCCGACTATCG | ||
X0-st4 | 5’BiotinCGATAGTCGGATCCTCTAGACAGCTCCATGTAGCAAGGCACTGGTAGAATTCGGCAGCGT | ||
X0-Adj | X0-st1, X0-st2, X0-st3 & X0-st4 | ||
X0In_st2 | GGGTGAACCTGCAGGTGGGCAAAGATGTCCATCTGTTGTAATCGTCAAGCTTTATGCCGT | ||
X0In_st4 | 5’BiotinCGATAGTCGGATCCTCTAGACAGCTCCATGTAGCAAGGCA/iCy3/TGGTAGAATTCGGCAGCGT | ||
Nk-X0 | X0-st1, X0-st2, X0-nk3a, X0-nk3b & X0-st4 | ||
X0-nk3a | ACGGCATAAAGCTTGACGA/iAF647-dT/TACAACAGATC | ||
X0-nk3b | ATGGAGCTGTCTAGAGGATCCGACTATCG |
Table 2: SMFRET and EMSA HJ substrates. The list of oligonucleotides used for the preparation of the fluorescently labeled HJs for smFRET and EMSA. The oligos were commercially obtained. The fluorescently labeled oligos were HPLC-purified and, when possible, oligos of ≥60 bp were PAGE-purified.
In this study, different smFRET techniques were implemented to determine the kinetics of HJ resolution by GEN130. Similar smFRET approaches were used to follow the double-flap DNA conformational requirement and cleavage by the DNA replication and repair flap endonuclease 142,43,44. Here, critical steps in this protocol are discussed. The silanization reaction should be free from any trace of humidity. The pegylation solution should be applied rapidly to the silanized glass once PEG is dissolved to avoid hydrolysis. In the multi-channel flow cell, any trapped air in the adhesive sheet should be removed to avoid leakage between neighboring channels. The PCA solution should be freshly prepared since it oxidizes over time. The addition of 10 N NaOH should be dropwise, with vortexing in between. The fluorescence background in the coverslip should be minimal before flowing the fluorescently labeled HJ. The imaging in the flow cell should be performed in one direction to avoid imaging bleached areas. In ALEX experiments, the power of the red laser should be reduced to avoid rapid bleaching of the acceptor. In the time-lapse experiments, the cycle time has to be shorter than the fastest event.
smFRET is a sensitive technique that can provide valuable real-time insights in biomolecular reactions. However, this method has several technical challenges, among which is achieving measurable change in FRET during the biochemical reaction. This is necessary to obtain well-separated features in the histograms and distinguishable states in the time-traces. In many cases, smFRET requires careful design of the substrates, selection of the fluorophore pairs and their positions, and amplification of FRET changes in the DNA substrate because of the little structural changes in the substrate45. Another approach for performing FRET is to use labeled proteins46. The observation window in FRET is limited by the stability of the acceptor such as Cy5 or Alexa Fluor 647 which tends to bleach more rapidly than the donor (Cy3 in this case). Therefore, FRET requires a continuous search for stable fluorophores to extend the experiment duration and efforts to develop oxygen scavenging systems to prolong the fluorescence signal and maximize the signal-to-noise ratio47,48.
Among the tips for troubleshooting in smFRET is balancing the several parameters involved in imaging such as the laser power, exposure time, cycle time, and number of cycles to maximize the fluorescence emission, prolong the experiment duration, and achieve appropriate sampling intervals for the enzyme dynamics. Longer observation times and minimal effects from photobleaching are essential to obtain high fidelity dwell time distributions that represent the enzyme dynamics. ALEX generates better histograms since this method is subjected to lower contributions from photobleached particles compared to single-color FRET. However, the temporal resolution in ALEX is lower than that in single-color FRET.
Finally, smFRET’s emphasis on detecting conformational/structural changes in individual molecules in real-time bridges the gap between high resolution structural techniques (i.e., X-ray crystallography, nuclear magnetic resonance, electron microscopy), which provides atomic resolution structural details under static conditions and bulk methods that yield the ensemble average of a measurable property. In many aspects, smFRET has proven to be a powerful technique for studying biological systems in real-time.
The authors have nothing to disclose.
This work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award (CRG3) to S. M. H.
(±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) | Sigma-Aldrich | 238813 | |
0.1 M sodium bicarbonate buffer | Fisher | 144-55-8 | |
10 % Novex Tris-Borate-EDTA gel | Thermo Fisher Scientific | EC6275BOX | |
100 X TIRF objective | Olympus | NAPO 1.49 | |
3,4-dihroxybenzoic acid (PCA) | Sigma-Aldrich | P5630 | |
3-aminopropyltriethoxysilane (APTES) | Sigma-Aldrich | 741442 | |
6% Novex Tris-Borate-EDTA gel | Thermo Fisher Scientific | EC6265BOX | |
Adhesive sheet | Grace bio-labs | SA-S-1L | |
Benchtop refrigerated centrifuge | Eppendorf | Z605212 | |
Biotin-PEG | Laysan Bio | Biotin-PEG-SVA 5000 | |
Bovine Serum Albumin (BSA) | New England Biolabs | B9001S | |
Calcium Chloride Dihydrate | Sigma-Aldrich | 31307 | |
cation exchange column | GE healthcare | MonoS (4.6/100) | |
Cell distruptor | Constant Cell Disruption System | TS5/40/CE/GA | |
Coomassie Brilliant Blue | MP Biomedicals | 808274 | |
Cy3 emission filter | Chroma | HQ600/40M-25 | |
Cy5/Alexa Fluor 647 emission filter | Chroma | HQ700/40M-25 | |
Dichroic for DV2 filter cube | Photometrics | 630dcxr-18×26 | |
Dithiothreitol (DTT) | Thermo Scientific | R0861 | |
Drill | Dremel | 200-1/21 | |
Electronic cutter | Copam | CP-2500 | |
EMCCD camera | Hamamatsu | C9100-13 | |
Epoxy glue | Devcon | 14250 | |
FPLC Aktapurifier UPC 10 | GE Healthcare | 28406268 | |
GelQuant.NET software | biochemlabsolutions.com | Version 1.8.2 | |
GEN1 entry vector | Harvard plasmid repository | HSCD00399935 | |
Glycerol | Sigma Life Science | G5516 | |
green laser (emission 532 nm) | Coherent | Compass 315M-100 | |
Heparin column | GE healthcare | HiTrap Heparin column | |
HEPES | BDH | BDH4162 | |
Image splitter | Photometrics | Dualview (DV2) | |
Imidazole | Sigma-Aldrich | I2399 | |
Inverted microscope | Olympus | IX81 | |
Isopropyl-ß-D-thiogalactoside (IPTG) | Goldbio. | 12481C100 | |
Laser scanner | GE healthcare | Typhoon Trio | |
LB Broth | Fisher Scientific | BP1426-500 | |
Long pass 532nm filter | Semrock | LPD02-532RU-25 | |
Magnesium Chloride | Sigma Life Science | M8266 | |
mPEG | Laysan Bio | mPEG-SVA 5000 | |
Neutravidin | Pierce | 31000 | |
Ni-NTA column | GE healthcare | HisTrap FF | |
NuPAGE 10% Bis-Tris gels | Novex Life technologies | NP0301BOX | |
NuPAGE 10% Bis-Tris Protein Gels | Thermo Fisher Scientific | NP0302PK2 | |
Origin software | OriginLab Corporation | Version 8.5 | |
Phenylmethylsulfonyl fluoride (PMSF) | Alexis Biochemicals | 270-184-G025 | |
Phosphate-buffered saline | GIBCO | 14190 | |
Polyethylene Tubing (I.D. 0.76 mm O.D. 1.22mm) | Fisher (Becton Dickinson) | 427416 | |
Protocatechuate 3,4-dioxygenase (3,4-PCD) | Sigma-Aldrich | P8279-25UN | |
Quad-band dichroic | Chroma Inc | Z405/488/532/640rpc | |
red laser (emission 640 nm) | Coherent | Cube 640 100C | |
Sodium Chloride | Fisher Chemical | S271 | |
Sorvall RC-6 plus centrifuge | Thermo Fisher Scientific | 46910 | |
Spectrophotometer | Thermo Fisher Scientific | Nanodrop 2000 | |
Syringe pump | Harvard Apparatus | 70-3007 | |
Teflon tweezers | Rubis | K35A | |
Tris Base | Promega | H5135 | |
Ultracentrifuge | Beckman Coulter | Optima L-90K | |
Ultrafiltration membrane | Millipore | UFC90300 |