We have developed a FRET mapping methodology that allows the identification and characterization of ligand binding sites, subunit orientation, conformational changes associated with ligand binding, and dynamic motions of proteins. An advantage of this technique is it can be performed in solution and the molecules can move around freely. The distances measured by this technique are appropriate for biological systems.
FRET is broadly applicable to many systems. Mapping enhances the monitoring of structural and dynamic changes in any biomolecular system, and is particularly effective if three-dimensional structural information exists. After purifying the protein, the hardest part is labeling with high efficiency, and removing the free dye.
For the experiments, it helps to have as little free dye as possible. To begin, choose labeling sites within 25 to 75 angstrom of the punitive binding site, and in relatively static regions of the protein. The distance will determine the specific FRET dye pair to be used.
Locate the labeling sites in protein regions that are relatively distinct from each other. So the sites describe the vertices of a triangle, with the punitive binding site located in the center. To measure the R0 values, depending upon the desired cuvette size and volume, prepare two protein samples at the same concentration of total protein.
One with the protein labeled with the donor dye only, and one with the protein labeled with the acceptor dye only. Turn on the fluorometer and open the spectral acquisition and analysis program in the FluorEssence software, if using a spectrofluorometer. Click on the red M to connect the computer to the instrument and choose Emission spectra.
Enter scan parameters using the collect experiment menu item, such as excitation wavelength, the range for emission scan, temperature, and sample change your position. Click on RTC and optimized instrument settings as described in the manuscript, by monitoring the fluorescence emission at the peak using an excitation wavelength set at the dye's absorption maximum. Place the donor labeled protein sample in the sample holder, and click run to generate an emission scan of the protein labeled with the donor dye only, by exciting the sample at the dye absorption maximum, and scanning over the emission peak.
Establish a baseline for the scan by extending the scan at 25 to 50 nanometers past the end of the peak. Measure the quantum yield of the donor only protein by performing absorptions and fluorescence measurements on samples of different concentrations, as described in the text manuscript. Prepare donor only protein, acceptor only protein, and donor-acceptor protein samples, at the same concentration.
Obtain the donor only scan to generate fluorescence emission spectra. Excite the solution at the donor dye absorption maximum, and scan over the donor and acceptor emission peaks. Either exchange the sample to the acceptor only protein, or change the sample change your position to the cuvette containing the acceptor only protein.
Obtain an emission scan of the protein labeled with the acceptor dye only. Excite the sample at the donor excitation wavelength. Exchange the sample to the donor-acceptor protein sample or change the sample change your position to the cuvette containing the donor-acceptor labeled protein.
Obtain an emission scan of the donor-acceptor protein sample using the same settings. For all spectra, correct background fluorescence by subtracting the background counts measured at the end of the scan. Use a 3D graphical viewing program such as PyMOL to map the distances onto the structure, by directly entering the commands from the script into the command window with the appropriate distance information.
Generate a shell for each distance measured, and the associated error. Map the position through the intersection of the different shells. The signal peptide binding site was mapped using three different locations on SecA and SecYEG, and four different locations on the signal peptide.
FRET experiments between different regions of the pre-protein and three distinct locations on the SecA and SecYEG proteins map the pre-protein binding site and orientation. The punitive binding site of the signal peptide was previously identified as the two-helix finger, and the SecA C-terminal tail suggested an orientation parallel to the two-helix finger. Steady state FRET spectra between SecA37, and PhoA2, and PhoA22, were obtained.
Reduction in the donor intensity indicated that greater energy transferred from PhoA22. Relative to the PhoA2 site, which is located further away from SecA37. Time resolved fluorescence decay spectra demonstrated that the donor-acceptor complex has a shorter homogenous decay consistent with energy transfer associated with one distance.
FRET distance shells constructed between the SecA PhoA2 residue, and the triangulated sites, demonstrated that each shell intersects with the two-helix finger, the assumed binding site, which is shown in green. The intersected area is smaller than each FRET shell and includes a large contribution from the helical scaffold. FRET distance shells determined between the PhoA2 residue and the labeled sites, describe a smaller area located closer to the tip of the two-helix finger and the mouth of the SecYEG channel.
This intersected area is situated at the opposite end of the two-helix finger from PhoA2. Important things to consider include proper selection of the labeling sites to triangulate the area of interest, to measure the quantum yields for each site, and remove all the free dye. This method aligns with structural information obtained with methods like NMR or X-ray crystallography.
When the FRET results are put into a structural context it can enhance the existing information.
