Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

The overall goal of the following experiment is to measure the confirmational changes in membrane proteins in their physiological state. This is achieved by site-specific labeling of membrane proteins to measure their LRE lifetime, and thus the distance between the two sites. As a second step, a ligand is added inducing a confirmational change that can be measured.

Next, the appropriate protease is added to cleave one of the floor fours and obtain the background signal. Ultimately, the change in the fluorescence lifetime from the resulting residents energy transfer can be measured to measure the confirmational changes that occur in the protein structure. The main advantage of this technique over existing methods like X extra crystallography, is that with this method we can measure the dynamic changes of proteins in their physiological state.

Though this method can provide insights into inotropic lide receptors or asay sensing ion channels, it can also be applied to other types of proteins like NICO receptors. We are gonna show the technique of luminess and stress and energy transfer, particularly in the context of studying confirmational changes of membrane proteins in mammalian cells. My students, Dino and Swara Swami, will be demonstrating the procedure Before starting the experiment.

It is important to choose labeling sites that are able to reflect the possible conformational changes within the protein. If possible, use a crystal structure of the protein or of a homologous protein to help make this determination. Choose residues such that the side chains of the selected residues are surface exposed and accessible to the Fluor fours so that the protein can be labeled.

Include a protease cleavage site that can specifically cleave off one of the cystines from the protein if the protein sequence allows for it. Introduce a site by conservatively mutating the protein to have a thrombin or factor 10, a sequence near the introduced cystine and accessible to protease cleavage. Choose a site such that upon cleavage one of the introduced cystines dissociates from the rest of the protein.

In certain cases, this may require two cleavage sites flanking a mutated cystine. Select the floor fours based on the expected distance range being measured, such that the range is between 0.5 and one times the R zero of the floor four pair. This allows for an easier subtraction of the background, which typically has a much longer lifetime for a range of around 35 angstroms and appropriate floor four pair would be tur chelate as the donor and Alexa 5 94 as the acceptor with an R zero of 53 angstroms.

Once the protein of interest has been selected, detach the HEK cells by simply pipetting buffer against the bottom of the dish. Then spin down the cells and resuspend the pellet in three milliliters of extracellular buffer. Next, incubate the cells in the donor and accept their floor.

Fours in equimolar amounts up to a final concentration of 100 to 300 nanomolar on a rotator for one hour at room temperature. After labeling, wash the cells three to four times with extracellular buffer to remove any unbound fluor and resuspend the pellet in two milliliters of fresh extracellular buffer. To measure the lre of the cells, transfer the cell suspension into a quartz vete with a minimum volume of one milliliter, and set the excitation wavelength of the photoluminescent spectrometer to the absorbance range of the donor fluoro four.

Set the emission wavelength appropriately. Then set the length of the emission detection to be at least three times the expected LRE lifetime to ensure that a long lifetime component will not be missed and perform at least three scans of 99 sweeps to ensure consistent results. Save the results as a text file, and then to measure the confirmational changes of a protein in response to the addition of a ligand.

Add the ligand and perform at least three scans of 99 sweeps each on the same sample under this new condition. Then add up to five units of the appropriate protease. Continually scanning until the cleavage is complete and no further change is seen in the lifetime for three successive scans, about two to three hours to analyze the LRE data.

Begin by setting the average values as a line graph with the fluorescence intensity on the Y axis and the lifetime in microseconds on the x axis to create a curve that represents the lifetime of the sensitized emission of the acceptor. Change the Y axis of the plot to a log scale. Then under the plot menu, open the layer control dialogue box and transfer the data containing the background mean into the list of layer contents.

Next, under the math menu, use the simple math function to multiply or divide the background data as needed until the tail end of the background overlaps with the tail end of the raw data, leaving the background signal present from before and after the protein cleavage. Then use the simple math function again to subtract the aligned background signal from the initial raw LRE signal and fit the data to an exponential decay. Set the start boundary for fitting to begin after the end of the laser pulse.

Then in the select fitting function dialogue box, select exponential decay so as to fit the lifetime to the equation for a single exponential decay. Where Y represents the fluorescence intensity, Y zero represents the background intensity due to noise from the system. A one is the signal intensity, T is the fluorescence lifetime, X is the time, and X zero is the time offset.

Finally, fix X zero to zero. Start the fitting function and fit the data using the lifetimes obtained from the data. Use the forestry equation to calculate the distance between the floor fours.

A successful LRE measurement with a lahane donor should have a donor only lifetime in the millisecond time range. When measuring confirmational changes, the specific LRE signal should show a change in the lifetime outside of the error of the measurement, which can then be calculated by the propagation of the errors associated with the fit of the lifetimes. The lifetime of sensitized LRE emission for the protein labeled with both the donor and the accepter will be notably shorter with a lifetime in the millisecond range.

After background, subtraction, protease cleavage results in an increase in lifetime that should become stable over time, indicating that the protease cleavage is complete. If the LRE signal comes from only one set of sites, the resulting emission lifetime after subtraction of the background should give a single exponential lifetime. Once mastered, this technique can be completed in four to five hours if performed properly.

While attempting this procedure, it's important to monitor the parameters, wavelengths, and steroid speed After its development. This technique has paved the way for studying conformational changes in membrane proteins in their neurophysiological state.