Fluorescence Fluctuation Spectroscopy to Study Protein Homo-Oligomerization

Overview

This video demonstrates the technique of fluorescence fluctuation spectroscopy (FFS) to study protein homo-oligomerization. Fluorescently-labeled proteins in a sample are dimerized using an agent when studying protein oligomerization using FFS. Using a confocal microscope, as the proteins move in and out of the small observation volume, the analysis of the fluctuations in the brightness of the fluorescent molecules is performed to determine the oligomeric state of the proteins.

Protocol

1. FKBP12 F36V -mVenus Purification

1. Transform (DE3) pLysS cells with pET22b vector containing monomerized human FKBP12F36V12 and N-terminal His6 and mVenus tags (vector available on request). Plate cells onto LB agar supplemented with 50 µg/mL Ampicillin and 34 µg/mL Chloramphenicol.
2. Transfer transformed colonies into 100 mL LB starter culture and grow for 16 - 20 hours at 37 °C with shaking.
3. Dilute dense starter culture (OD600 >1) 1:100 in LB medium (2 x 500 mL batches) and grow for 2 - 3 hours to OD600 = 0.6 - 0.8 (37 °C, 200 rpm ).
4. Cool cultures on ice. Induce with 250 µM IPTG and grow for 16 - 20 hours at 21 °C, 200 rpm.
5. Harvest cells by centrifugation at 2,000 x g for 20 minutes.
6. Resuspend pellet in 40 mL of IMAC buffer A (20 mM sodium phosphate pH 7.5, 500 mM NaCl, 3 mM imidazole, 1 mM β-mercaptoethanol) supplemented with EDTA-free protease inhibitors (1 tablet per cell pellet).
7. Sonicate cells (500 Watts, 20 kHz, 40% amplitude, 9 s on, 11 s off for 15 min) on ice and harvest soluble material by centrifugation at 20,000 x g.
8. Transfer soluble lysate to a conical flask and add 2 mL of resin (see Table of Materials). Incubate for 1 hour with 105 rpm rotation
   NOTE: Nickel sepharose may also be used for this IMAC step.
9. Harvest resin and wash with 250 mL of IMAC buffer A followed by 500 mL of IMAC buffer B (20 mM sodium phosphate pH 7.5, 500 mM NaCl, 7 mM imidazole, 1 mM β-mercaptoethanol).
   NOTE: Increase to 50 mM imidazole if using Nickel sepharose resin.
10. Elute His6-tagged protein using IMAC buffer C (20 mM sodium phosphate pH 7.5, 500 mM NaCl, 300 mM imidazole, 1 mM β-mercaptoethanol).
11. Inject onto a size exclusion column (see Table of Materials) equilibrated in 10 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM DTT. FKBP12F36V has its peak elution at 87.71 mL on the column we used.
12. Assess purity via SDS-PAGE and pool and concentrate as required.

2. Preparation of Multiwell Plate Array

1. Thaw the purified FKBP12F36V (or labeled protein of interest) from -80 °C.
2. Prepare a solution of 100 nM purified FKBP12F36V (medium, 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT). Sonicate and centrifuge (quick spin of 13,000 rpm) to prevent the formation of aggregates.
3. Pipette 100 - 200 µL into an 8-well observation chamber with a glass bottom.
4. Add the BB dimerizer to final concentrations of 10, 20, 40, 80, 100, 150, 300, and 500 nM.
5. As a reference, prepare a solution of 100 nM of mVenus alone to evaluate potential aggregation and precipitation effects and recover a brightness value for the monomer with the same acquisition settings.

3. Calibration-free Confocal Acquisition

1. Start the confocal system (Figure 1). Any light scanning microscope confocal system equipped with digital detectors or well-characterized analog detectors, and capable of keeping a constant dwell time for every pixel acquired would work.
2. Set the excitation beam path:
   1. Turn on the 514 nm laser and set it at 20 - 100 nW Power at the exit of the objective (for FKBP12F36V-mVenus).
   2. Select the 63X1.4NA objective or a collar correction water immersion objective designed for FCS.
   3. Turn on one HyD, APD, or calibrated PMT detector. Detectors capable of photon-counting are preferable, as in this case, calculation of S, offset and σ₀² are unnecessary.
   4. Select the emission window from 520 - 560 nm
   5. Set the pinhole at 1 Airy unit for the corresponding emission ~545 nm.
   6. Set the acquisition mode to 16 x 16 pixels
   7. Set the pixel dwell time t_dwell such that it satisfies t_frame >> T_D >> t_dwell, where T_D is the residence time of the diffusing protein and t_frame is the frame rate. This corresponded to setting the dwell time to ~13 µs.
      Note: Some commercial manufacturers had scanners that were not keeping the dwell time per pixel constant. This constancy is crucial for the method to work.
   8. Set the pixel size at ~120 nm.
   9. Select the xyt acquisition mode and select the number of frames to be acquired per acquisition and well (for example 5,000).
   10. If the system is equipped with a high-throughput mode, introduce the coordinates of each well and the number of acquisitions per well to automate the process.
       Note: Be careful to ensure the presence of a water dispenser if using an immersion objective.
   11. If the system is equipped with a perfusion system, load the BB solution and program the perfusion to start right after the 5000^th frame to evaluate the kinetics of dimerization while acquiring e.g., 10,000 images.
3. Add a drop of oil into the oil immersion objective/water if utilizing a collar correction water immersion objective designed for FCS.
4. Mount the 8-well observation chamber on the stage.
5. Select the correct well and focus on the solution.
   Note: IMPORTANT: Avoid focusing close to the bottom glass to avoid reflection and scattering. When focusing deeper into the solution, disconnect the automated focus option.
6. Start the acquisition and save the resulting stack of images in TIFF format.

4. Detrend and Brightness Analysis using the R Package nandb

1. As a preprocessing quality check, use ImageJ to take a look at the images and recover the intensity profile, as shown in Figure 2a. This is useful to determine whether or not too much photobleaching has occurred. If there is too much bleaching, the data is not suitable for further analysis.
   Note: ImageJ can also be useful to convert images to TIFF from commercial formats. The nandb software described below can only work with TIFF files.
2. Download and install R and RStudio. It is best to download and install R first, then RStudio.
   NOTE: What follows is a description of how to use the nandb R package. Knowledge of the R language is not required to use nandb, however, it will make life easier.
3. Install the nandb package.
   1. Open RStudio and in the console, type install.packages("nandb") and wait for the installation.
4. Get to know nandb
   1. Review the manual.
   2. Review the built-in RStudio help for various functions. The most likely function to be used will be using will be brightness(). View the help file for this function by typing ? brightness() at the console.
5. Calculate brightness
   1. Say one has an image file on the desktop called img001.tif (note that `nandb` only works with TIFF files). One can calculate the brightness of that image:
      b <- brightness("~/Desktop/img001.tif", tau = "auto")
      1. This assigns the brightness of the image to the variable b in R. The tau = "auto" ensures that the image is correctly detrended prior to brightness calculation. The most common thing to do from here is to calculate the mean or median brightness of the image. One can do this by typing mean(b) or median(b). One can also write the brightness image on the desktop using
         ijtiff::write_tif(b, "~/Desktop/whatever_img_name")
   2. Say one has a folder full of images images_folder on the desktop and one needs to calculate the brightnesses of these images and write the brightness images as TIFF files. Then see ?brightness_folder(). This function processes a whole folder all at once:
      brightness_folder("~/Desktop/images_folder", tau = "auto")
      This is particularly good for those who have software they prefer to R because all of the files are processed in one single command, and then one can go on working with the output brightness TIFF images in their chosen software, be it ImageJ, Python, or something else.

Representative Results

Figure 1. Application of N&B to detect protein monomer-dimer transitions in solution. (a) Simplified optical path of a laser scanning microscope (LSM) equipped with a laser source (set at 514 nm in the case of mVenus labeled proteins) directed (blue arrows) toward an immersion objective (in our case a 63X1.4NA oil) illuminating a 100 nM solution of FKBP12F36V-mVenus solution. The emission fluorescence (green arrows) passes through a dichroic mirror and is directed toward a bandpass filter that cleans the emission light, and a pinhole set at 1 Airy unit situated right before a point digital detector capable of photon counting. (b) A confocal volume of illumination is scanned through 16 x 16 pixels illuminating single FKBP12F36V-mVenus molecules that enter and exit the Gaussian shape confocal volume producing an array of fluorescence intensity fluctuations. (c) Image series acquired over time

Figure 2. Automatic detrending is needed to accurately measure a population of monomers in solution. (a) A stack of 5000 16 x 16-pixel images was acquired as described in Protocol section. The intensity of the first frame is shown together with the average time-resolved intensity profile, which shows long-term fluctuations that might be related to bleaching and other solvent and/or photophysic effects. Whatever the cause for these long-term fluctuations, they impact the brightness calculations and hence require detrending. Without automatic detrend, one gets B = 1.026, whereas after automatic detrending, B = 1.005. Also, the brightness without (left panels, second row) and with (right panels, second row) smooth filtering is shown. (b) The same data presented in (a) was detrended and the results in terms of intensity and brightness shown.

Materials

Name	Company	Catalog Number	Comments
RosettaTM (DE3) pLysS cells	Novagen	70956-3
Ampicillin	Sigma Aldrich	PubChem Substance ID 329824407
Chloramphenicol	Sigma Aldrich	PubChem Substance ID: 24892250
LB starter culture	QIAGEN
LB medium	QIAGEN	https://www.sigmaaldrich.com/content/dam/sigma-aldrich/head/search/external-link-icon.gif
IPTG	Sigma Aldrich	PubChem Substance ID 329815691
IMAC buffer	Medicago	09-1010-10
EDTA-free protease inhibitors	Sigma Aldrich	11873580001
TALON resin	Clonetech
Nickel sepharose	GE Healthcare
S200 16/60 column	GE Healthcare
Glass bottom 8-well observation dish	Ibidi	80827