Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

The overall goal of this procedure is to study the dynamics of complex protein systems in a quantitative way with a focus on correlated interactions such as cooperativity. This method can help answer key questions in the field of quantitative biology where protein complexes and their dynamic interactions are of central importance. The main advantage of this technique is that it allows dynamic multicolor single molecule FRET to be applied to biologically relevant systems.

Prior to assembling the flow chamber cut a flow channel in a 40 micron thick transparent adhesive film spray quick fixing adhesive on the non adhesive side of the film and allow the adhesive to dry. To begin flow chamber assembly obtain a three millimeter thick PEG biotin PEG functionalized fused quartz slide with inlet and outlet holes. Apply the spray adhesive coated side of the ceiling film to the functionalized side of the slide aligning the channel with the inlet and outlet hooks.

Heat the slide to 80 degrees Celsius on a hot plate for one minute. Press the film onto the slide for 30 seconds using an extra microscope slide to distribute the pressure evenly. And then allow the assembly to cool for one minute.

Peel off the protective sheet covering the adhesive. Place a cover slip on the exposed adhesive to form the flow chamber. Heat the chamber to 80 degrees Celsius for one minute.

Press the cover slip onto the adhesive for 30 seconds to seal the chamber and allow the assembled chamber to cool. Apply a drop of glycerol to the prism face before placing the flow chamber over the prism. Mount the flow chamber in a holder for a multi color prism type TIRF microscope.

Connect the inlet and outlet tubing when finished. To begin configuring the instrument for the three color measurement open the electron multiplying CCD camera software set the EMCCD sensor to the lowest possible temperature. Set the camera vertical shift speed to 3.3 microseconds, choose normal vertical clock voltage, set the horizontal read out rate to 17 Millihertz at 16 bit, the pre-amplification gain to three and the electron multiplier gain level to 1000.

Choose external acquisition triggering. Set the exposure time to 70 milliseconds. And the movie recording duration to 750 acquisition cycles.

Create a new folder for the measurement files. Enable auto saving in the EMCCD software and set the movie frame file format to TIFF. Set the auto save file path to the newly created folder.

Then open the software controlling the acousto-optical tunable filter, the laser control software, and the trigger software synchronizing the lasers the AOTF, the optical shutters, and the cameras. Use the AOTF to adjust the laser power to about three milliwatts before entering the prism. Load the triggering pattern that synchronizes all devices for the alternating laser excitation then mount the sample holder in the instrument.

Place the end of the inlet tubing in a microcentrifuge tube containing the experiment buffer. Connect the outlet tubing to a syringe pump set to withdraw mode. Flush the chamber with 150 microliters of buffer.

Focus the CCD camera on the functionalized interface align the excitation beams and bleach fluorescent contaminants. Then flow 300 microliters of a 0.25 milligram per milliliter solution of deglycosylated avidin and buffer into the chamber and incubate for one minute. Flush unbound deglycosylated avidin from the chamber with buffer.

Then flow 300 microliters of buffer with a BSA concentration of 0.5 milligrams per milliliter through the chamber to block surface functionalization defects. Next, load the flow chamber with 150 microliter portions of biotinylated fluorescently labeled Hsp90 in BSA containing buffer at increasing Hsp90 concentrations until sufficient surface density is achieved. Wash unbound protein from the chamber with 300 microliters of BSA containing buffer.

Load 150 microliters of a 25 nanomolar solution of labeled AMP-PMP and BSA containing buffer into the chamber and incubate for five minutes. Repeat the loading and incubation once. Then use a Piezo stepper to move the sample chamber perpendicular to the excitation beam to change the field of view as desired.

Adjust the image focus if necessary. Start the camera recordings in the camera software by clicking the take signal button as shown. Initiate the excitation acquisition cycles in the trigger software to begin data acquisition.

To begin data analysis, open the analysis software and import the recorded movies of the two cameras. Click find traces to determine the fluorescence intensity traces corresponding to potential single molecules. For each molecule, calculate the sum of all traces of that spot with the same excitation color.

Evaluate the intensity profiles in all channels for a roughly flat plateau in the joint raw intensity and a single bleaching step for all excitation colors. Look for anti correlated behavior in the appropriate detection channels and the occurrence of red fluorescence in the trace. If all criteria are met save the fluorescence traces for further analysis.

Only the indicated molecule fulfills the criteria in this example. Exclude traces that do not have flat plateaus, that show blinking events rather than anti correlation or that have multiple bleaching steps. Trace selection is a critical step in all single molecule techniques.

Only traces that fulfill well defined criteria should be selected. Here these criteria are anti correlation, single bleaching steps, and flat plateaus. Next, display the saved molecules one after another and one set of intensity traces select a time interval in which all floor fours are already bleached.

Calculate the beam background intensity for this time interval then select the FRET efficiency range in which both dyes on Hsp90 are present being sure to exclude traces with blinking events. Calculate the partial fluorescence traces. Exclude molecules with a low signal to noise ratio in the traces.

Next, generate bind 2D projections of the partial fluorescence data and start the relative population calculation tool. Click Init. Draw a polygon around the peak of interest.

And click count to calculate the relative population for that peak. Generate a 3D histogram of the partial fluorescence data and normalize the histogram. Excess the initial parameters for a 3D Gaussian fit and add the state populations to the end of the parameter vector.

Fit the data and display the results. After defining the position and width of each state in the 3D partial fluorescent space initialize the Hidden Markov Model interface. Select the appropriate number of states, the number of dimensions of the input signal, and the input type.

Optimize the HMM parameters to determine the maximum likelihood estimators for the transition probabilities. Repeat the population selection, fitting, and HMM optimization for data subsets. Finally calculate the confidence interval for the transition probabilities and collapse the conformational states to simplify the data for further analysis.

The conformational states of the Hsp90 protein were studied with three color single molecule FRET in the presence of the labeled reporter nucleotide AMP-PMP. Five conformational states were distinguishable by fluorescence intensities with four of those states being functionally distinct. Three color single molecule FRET resulted in the partial fluorescence data spanning a 3D space.

2D projections were used to help separate the states as all states theoretically expected under the experiment conditions are distinguishable by their partial florescence in 2D projections. The relative populations of these states were determined from the 2D projections and were used as constraints for 3D Gaussian fits. Subsequent ensemble 3D HMM optimization and modeling provided the extracted state transition probabilities.

Confidence intervals were calculated for each extracted rate constant. Repeating the experiments in the presence of additional 250 micromolar unlabeled AMP-PMP elucidated the influence of the binding of one nucleotide on the second binding site in the Hsp90 dimer. Collapsing the bound and unbound conformations allowed the calculation of the average dwell time for the fluorescent AMP-PMP dissociation from Hsp90 in the presence and absence of additional unlabeled AMP-PMP.

After watching this video you should have a good understanding of how to extract kinetic information from a dynamic protein system using multicolor FRET and a TIRF microscope. This technique paves the way for researchers in life sciences to determine correlated interactions in multi protein systems. This is important for a deeper understanding of fundamental regulatory mechanisms such as cooperativity.