Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

This research aims to study the local flexibility and dynamics of biomolecules at the single molecule level, using time-resolved fluorescence anisotropy in confocal microscopy mode. Single molecule detection and localization have enabled the observation of biomolecules, yet capturing their dynamics require other approaches. Time-resolved fluorescence methods help fill that gap.

Common tools for experimentally studying biomolecular dynamics including single molecule fret and NMR. At the single molecule level, various analysis, process, the photon by photon fluorescent emission, These include time-resolved analysis and burst variance analysis.

Using multiple fluorophores for smFRET experiments can introduce complexities in the experimental design and in making samples of good enough quality. Using a single label and fluorescence anisotropy solves some of these complexities.

We found that the monomeric form of the forkhead domain of the human FoxP1 behaves as a disordered protein and increases its folding population when it dimerizes.

[Instructor] To begin, filter the prepared standard buffer solution through a 0.22 micrometer pore-size filter. Then filter the standard buffer through a charcoal filter for single molecule acquisition. For the fluorescent probes, prepare small volume aliquots of the prepared BODIPY Fluorescent Pro. For calibration of the polarization resolved setup, first, turn on the parallel and perpendicular detector channels and power on the blue laser. Ensure that the laser power is set to 60 microwatts and open the time-correlated single-photon counting or TCSPC software control panel. Next mix one microliter of 100 nanomolar Rhodamine 110 with 49 microliters of distilled water. Add a drop of immersion liquid on top of the microscope objective lens. Place the cover glass onto the microscope objective and ensure that the water droplet is centered on the lens. Add 50 microliters of the diluted Rhodamine 110 solution to the center of the cover glass for calibration measurements. Adjust the image plane to be inside the solution and above the glass surface. Using the focus knob, locate the second bright focal point and adjust it to 1 1/2 turns. Now open the software in Time-Tagged Time-Resolved mode and click the START button. Record the count rate for 120 seconds. For background measurements, add 50 microliters of distilled water to the center of the cover glass and repeat acquisition. Record the photon count rate for 300 seconds and save the file as water.ptu. For additional background measurements, add 50 microliters of standard buffer to the center of the cover glass. Record the photon count rate for 300 seconds and save the file as standard_buffer.ptu. Now launch the Burst Integration Fluorescence Lifetime, BIFL, analysis software and click on Confirm the setup from the automatic window. Next click Get Parameters From File, then click OK. For selecting the measurement to analyze, click on Data Path Array, and select the appropriate dataset. Now load the water measurement water.ptu file for arrays, such as Green Scatter, the standard_buffer.ptu file for the Green background, and Rhodamine110.ptu for Green thick. under the Single Molecule selection parameters, Click Next, then Adjust to open a new popup window. Then press Threshold to modify the interphoton arrival time and choose single molecule event time for the mean interphoton arrival time. Now click minimum number to set the minimum number of photons per single molecule event. Then close the popup window by clicking Return and OK. Click on Color Fit parameters to adjust the initial fluorescence lifetime for green and other colors using fluorescence decay parameters. Modify, prompt, and delay values by adjusting the from and to values and close the popup window. Finally, press Save to process the ASCII files and save them in the selected folder. Transfer an overnight pre-inoculum of Escherichia coli C41 into 500 milliliters of LB medium with pre-added antibiotic at a 1:500 dilution ratio. Measure the absorbance of the culture at 600 nanometers to monitor the culture growth. When the optical density reaches between 0.5 and 0.7, add IPTG to a final concentration of 0.5 millimolar to induce protein expression, and incubate. Centrifugate the bacterial cells at 3,000 g for 20 minutes at four degrees Celsius when the culture reaches an optical density of 1.4 to 1.6. After discarding the supernatant, store the pellet at minus 20 degrees Celsius until use. Next, add 50 to 100 micromolar FoxP1 protein to a tenfold molar excess of either DTT or TCEP and incubate. Place a PD-10 desalting column into a 50 milliliter centrifuge tube using a column adapter for buffer exchange. Add five milliliters of PBS buffer to the column for equilibration and centrifuge it at 1,000 g for two minutes. Transfer the PD-10 column into a fresh 50 milliliter tube, and add two milliliters of PBS buffer into the column. Now load 500 microliters of FoxP1 protein into the column. Elute the column by centrifuging at 1,000 g for two minutes to collect the purified protein. Transfer the protein into a 10 kilodalton molecular weight cutoff ultra centrifugal filter. After centrifuging at 7,500 g for 10 minutes, collect the concentrated eluate. Measure the protein concentration. Then add BODIPY and incubate at four degrees Celsius for two hours on a rotator. Repeat buffer exchange and protein concentration. After determining the protein concentration, measure the degree of labeling using the formula. Add 495 microliters of ultrapure water and five microliters of Tween 20 into a well of a chamber slide, and mix well. After a 30-minute incubation at room temperature, wash the chamber gently with ultrapure water twice and dry it. To correct for background fluorescence, perform measurements of a buffer solution. Calibrate the relative sensitivity of the parallel and perpendicular detectors by acquiring data from a fast-rotating dye For single-molecule fluorescence anisotropy experiments on monomeric FoxP1, add 100 picomolar BODIPY labeled FoxP1 and 100 nano molar unlabeled FoxP1 to 500 microliters of standard buffer in a chamber slide. Begin the measurements, and check in the BIFL software that the number of bursts recorded within 30 seconds is between 60 and 90. If the sample is more concentrated, dilute it until the burst count falls within this range. Finally, analyze the measurements. The single-molecule fluorescence anisotropy lifetime analysis revealed a single distribution originating from two distinct rotational correlation times in the FoxP1 DNA binding domain, suggesting the coexistence of ordered and disordered structural ensembles. Burst variance analysis identified a subset of FoxP1 molecules exhibiting high anisotropy and excess variants, suggesting the presence of dynamic conformational changes. Photon distribution analysis differentiated between monomeric and dimeric FoxP1 states, showing distinct anisotropy distributions and exchange rates. Dimerization influenced local and global motions in FoxP1 with excess variance measurements indicating significant anisotropic changes at specific cysteine-labeled sites. Dynamic anisotropy analysis revealed that FoxP1 undergoes partial unfolding upon dimerization and DNA binding, altering the fraction of structured and disordered populations.