The classical method in which membrane proteins are extracted for their study is by simple solubilization in detergent. While this provides a simple and universal method, detergents have been shown to alter the native structure, function and activity of these sensitive proteins. Here we present the Peptidisc as a solution for stabilizing membrane proteins in detergent-free solution.
The Peptidisc system spontaneously adapts to the size, shape and typology of myriad membrane proteins. This stands in contrast to other membrane mimetics that often require tedious optimization. FhuA, an E.coli outer membrane protein, will be used as a model target protein to be reconstituted in this protocol.
Outer membrane vesicles will be isolated followed by the addition of detergent to solubilize the membrane proteins. The Peptidisc peptide is added to the solubilization and detergent is diluted away, spontaneously forming Peptidisc particles. These stabilized soluble particles are now amenable for numerous downstream applications.
This protocol will examine bio-layer interferometry as an application of the Peptidisc technology. This protocol will focus on the PeptiQuick reconstitution method, a useful technique that facilitates the simultaneous purification and reconstitution of membrane proteins. The PeptiQuick method of on-beads reconstitution is performed in a standard Nickel-NTA IMAC gravity column pre-equilibrated in detergent.
The solubilized membrane is loaded onto the resin and non-target proteins are washed away. After this peptide is added to the column and the detergent is diluted rapidly. The hydrophobic side of the Peptidisc peptide associates with the exposed hydrophobic regions.
The hydrophilic side of the peptide keeps the protein soluble in solution. Excess peptide is washed away. Imidazole is added to elute the reconstituted fury.
The Peptidisc scaffold may be functionalized to indirectly label memory proteins and thus open the door to a broader set of downstream analyses. Here we exploit the biotinylated peptide for attachment to streptavidin coated sensors for BLI analysis. Bio-layer interferometry is a powerful label-free technique for investigating biomolecular interactions in solution.
As well as simply demonstrating interactions, this technique gives a real-time kinetic readout and enables the calculation of a binding dissociation constant. Through this protocol, we eliminate the need for detergent during the BLI analysis, as this can alter the activity. Biolayer interferometry analyzes the interference passing of white light reflected from the sensor, or tip, and bound macro molecules during an interaction.
To measure the interactions, the sense tip is passed between solutions and the signal recorded at each step. Each tip then gives its own trace on a sensogram. First, a baseline of buffer only is established.
Second, the ligand is bound. In this case, FhuA and biotinylated Peptidiscs binds a streptavidin encoated tip. Next, the tip is washed in buffer to remove any unbound FhuA.
Now the tip is moved into a solution with a known concentration of analyte, here ColM. If an interaction occurs, this will give a binding curve. Finally, the tip is moved into buffer and the dissociation measured.
The observed rates for association and dissociation at each column concentration can be used to calculate the dissociation constant. Hexahistidine tagged FhuA is expressed in E-coli strain AW740 for 18 hours in amine media. The bacteria are harvested by centrifugation at 5, 000 G's 10 minutes at four degrees Celsius.
The resulting cell pellet is re-suspended in TSG buffer and then dounced to break up cellular clumps and ensure thorough lysis. TMSF is added to the re-suspended cells to a final concentration of one millimolar just prior to lysis. Cell lysis is achieved by three passages through either a microfluidizer at 15, 000 PSI or a french press at 8, 000 PSI.
The lysed cells are collected and a low-speed centrifugation at 5, 000 G's for 10 minutes at four degrees is performed. A supernatant from the slow-speed spin is loaded into a TI70 ultracentrifuge rotor and centrifuged at 200, 000 G's for 40 minutes at four degrees to pellet the crude E.coli membrane. Following ultracentrifugation, the supernatant is discarded and the crude membrane pellet is re-suspended in a minimal amount of TSG buffer.
A crude membrane is dounced to ensure its homogeneity. A Bradford Assay is performed to check the protein concentration of the re-suspended crude membrane. TSG buffer is used to dilute the crude membrane to approximately three milligrams per milliliter prior to solubilization.
Triton X-100 is added to the re-suspended crude membrane at a final concentration of 1%to selectively solubilize the bacterial inner membrane. Solubilization is performed for one hour at four degrees, with gentle rocking. The un-solubilized outer membrane is isolated by ultracentrifugation at 200, 000 G's for 40 minutes, at four degrees.
The resultant supernatant containing the solubilized inner membrane is discarded. While the pellet, containing the outer membrane, is re-suspended as before in TSG. LDAO is added to a final concentration of 1%to the re-suspended outer membrane, and solubilized for one hour at four degrees.
Finally, an ultracentrifugation is performed, as before, to pellet insoluble material. The resultant supernatant contains solubilized outer membrane, including our target, his-tagged FhuA, now ready for simultaneous IMAC purification and Peptidisc reconstitution. A Nickel-NTA IMAC gravity column is pre-equilibrated with two column volumes of IMAC wash buffer.
Imidazole is added at a final concentration of five millimolar to the solubilized outer membrane, diluted to 0.04%LDAO. The solubilized outer membrane is loaded onto the Nickel-NTA resin, and the flow through collected. This flow through is reloaded onto the resin to increase the resin binding of FhuA.
Following loading, the resin is washed with 250 milliliters of IMAC wash buffer and the first 50 milliliters collected. After washing, the wash buffer is drained to just above the height of the resin bed and the column stopcock closed. One milliliter of concentrated, 10 milligram per milliliter, Peptidisc peptide is added to the column.
Following the addition of concentrated peptide, 50 milliliters of dilute, one milligram per milliliter, Peptidisc peptide in TSG is added. The resin is stirred to re-suspend the beads into TSG, below the CMC of LDAO, thus forming Peptidisc particles. Following Peptidisc trapping, the resin is allowed to settle and the one milligram per milliliter peptide solution is drained through the resin.
The resin is washed with 50 milliliters TSG to remove excess Peptidisc peptide. Finally, the Peptidisc particles are diluted with 50 milliliters of 600 millimolar imidazole in TSG. One milliliter fractions are collected and 10 microliters of 0.5 molar EDTA is added, a key light leached nickel ions.
The start, flow through, wash, and eluded fractions are loaded onto a 12%SDS gel and electrophorezed for 30 minutes at 60 milliamps. The gel is stained and visualized on a gel scanner. The resultant gel shows depletion of FhuA in the flow through, and enrichment in the elution.
Minor contaminant bands are also observed in the eluded fractions. Fractions three through seven are selected for size exclusion chromatography to confirm FhuA Peptidisc solubility and deplete contaminants. A 30 Kilodome cut off centrifugal concentrator is used to concentrate fractions three through seven.
One milliliter of the pooled IMAC elusion fractions is injected onto the S200 column at a flow rate of 0.25 milliliters per minute in TSG buffer. One milliliter fractions are collected and a 12%SDS gel of the fractions is run. The relevant fractions are pooled and may now be used in downstream applications.
BLI was conducted in a ForteBio Octet RED96. This instrument moves BLI pins across a 96 well plate which is first set up by hand. All wells are filled to a final volume of 200 microliters.
Column one is loaded with kinetics buffer to allow the tips to equilibrate and form a baseline signal. Column two is loaded with a pre-determined concentration of the ligand and kinetics buffer. In this case, FhuA is the ligand, and is added to the concentration of 2.5 micrograms per milliliter.
The tip in row E will be used as reference, and is loaded with buffer only. Column three is loaded with kinetics buffer to wash excess FhuA from the tip. Twofold serial dilutions of the analyte, in this case ColM, is loaded from top to bottom in column four.
The highest of these concentrations is used for the reference row, to measure for non-specific binding of the analyte to the tip. Column five is loaded with buffer. Here, the ColM will dissociate, and the dissociation measured.
Once the plate is prepared, the sensor-tip tray and prepared plate are loaded into the BLI instrument. The BLI data acquisition software is opened and a new kinetic experiment is started. The Plate Definition tab is used to input the layout of the 96-well plate into the software.
Here the ligand FhuA is input as the load while the analyte ColM is input as the sample. The Assay Definition tab is used to define the length of time and plate rotation speed for each step in the experiment. Here we include a baseline step of 60 seconds, followed by a loading step of 250 seconds, a second baseline step of 300 seconds, an analyte association step of 450 seconds, and finally a dissociation step of 900 seconds.
These steps are individually assigned to each column in the 96-well plate by selecting the desired step and right-clicking the Add Assay on each column. The sensor assignment tab is used to ensure that the Octet instrument is taking BLI pins from their correct location in the sensor tray. The Review Experiment tab provides a final overview of the experiment, prior to executing.
BLI will now be carried out by the Octet RED. This will produce a raw data sensogram for analysis. The analysis is performed by first opening the Octet biodata analysis software.
The Data Selection tab is used to locate the experiment and check the experimental summary. The concentrations of the analyte ColM are input here. Next, the Processing tab is used to subtract the reference signal from the experimental data.
The baseline is defined to align the Y-axis as if its de-alloy rate was applied. Finally, Process Data is selected. The data is isolated for this experiment, and Fit Curves is selected.
A partial association and dissociation curve fitting is chosen. The Kd is calculated from this fitting. The size exclusion chromatogram is used to validate the ambute's reconstitution.
The chromatogram shows a single symmetrical peak, suggesting a monodisperse protein preparation. The peak elutes several milliliters after the void volume, indicating non-aggregated soluble Peptidisc particles. Protein aggregates will elute at the void volume, while excess detergents and peptide will elute following the main peak.
Increased washing of the formed Peptidiscs while still attached to the Nickel-NTA beads should deplete this latter peak. Protein aggregates are often a result of exposing memory proteins to detergent free solution prior to reconstitution. This last exclusion chromatogram is a useful diagnostic for troubleshooting one's reconstitution.
Following processing of the experimental data, a curve is fit to the sensogram. The accuracy of this fitting is essential for the calculation of the dissociation constant. The residual view plot describes the difference between the experimental data and the computational fitting.
This shows that for the four different column concentrations three of the curves fit well. However, for the highest concentration the curve does not produce a good fit. This suggests that at this higher concentration, there is heterogeneous binding of column to the pin.
This highest concentration signal is therefore not included from the kinetic analysis as per the ForteBio application notes. The remaining three curves are used for the kinetic analysis. A dissociation constant between FhuA and ColM, determined using the BLI and Peptidisc method, is consistent with constants determined by other methods.
Isothermal calorimetry and nanodiscs, and microscale thermoforesis and Peptidisc yielded dissociation constants not significantly different from our determined value. This consistency validates the Peptidisc BLI method for measuring interaction kinetics. All right, so, after watching this video, we hope that you have gained a practical understanding of the strength and caveat of the PeptiQuick methods.
We have found this method particularly useful for quantifying the FhuA ColM interaction in the complete absence of detergents. And we suspect the same workflow can be applied to characterize any other receptor-ligand interaction. Good luck with your experiments.
