The overall goal of this procedure is to investigate the FLX pump me AB from Pseudomonas aosa using a functional test based on the reconstitution of membrane proteins. In liposomes. This is accomplished by first over expressing and purifying membrane proteins, mixed B and bacterial Rodin in bacteria membrane proteins are then extracted from the membranes and pure purified by affinity chromatography.
The second step is to prepare liposomes by combining a dried lipid film with buffer containing purine, which leads to the formation of multilam vesicles. Following sonication, the liposomes are extruded to get a homogenous population of liposomes. Next detergent membrane proteins and then polystyrene beads are added to the liposome solution.
The hydrophobic tails of the detergent add absorb on the polystyrene beads, allowing the membrane proteins to then loosen the detergent belt and insert into the liposome membrane. The last step is the removal of non encapsulated purine By running the protea liposomes on a desalting column, large prot liposomes reach the bottom of the column much faster than non encapsulated purine, which remains strapped at the top of the column. Ultimately, protal liposomes are collected and their fluorescence is used to investigate Mex AB activity through the change in pH created upon illumination of bacterial opsin.
Rodin Effects pumps are transporters that promote antibiotic resistance due to the recognition and transport of a broad range of substrates from all antibiotic families, there is an urgent need for new and reliable in vitro activity tests for membrane protein effects.Transporters. Indeed, such assays would offer new insights in the molecular mechanisms of transport and help the pharmacological screening of new potential inhibitor candidates. We focus on the mix a mix, B-O-P-R-M, eFlex Perm from Pseudomona gin.
We present technical advices and method necessary to satisfy three requirements for designing an efficient in vitro functional test for mix a B ffl pump, including the constitution of the protein in a membrane, use closed compartment necessary for the vectorial transport of substrate, a fine shows of flues and die that avoids artifactual effect due to the adro nature of the substrate and the generation of a protein gradient needed for mix AB to function. The strength of our assay is the way we generate a reproducible T enable and reversible pro gradient to create a delta pH we call nstitute bacteria or VR with mix AB in a liposome membrane. BR is a membrane protein purified from the purple membrane of aob salinium allic Marin gram-negative, obligate aerobic aans.
Upon illumination, this protein actively pumps proteins. Therefore, if BR is constituted together with the protein in the liposome, elimination of the liposome will create a delta pH due to the br, which pumps proteins inside of the liposomes. Preparation of pure and stable protein material is of foremost importance.
Purification procedure of membrane protein makes B have been nicely described in a previously film J ovy publication, as well as in the text protocol accompanying this video where a description of the purification of a membrane protein mix A can also be found To prepare bacterial Rodin from Halabach Salinium grow halabach salinium cells under illumination at 37 degrees Celsius for 10 days. In a liquid growth medium, disrupt the cells by dialyzing against deionized water. Purify the purple membrane on a 30 to 40%sucrose gradient.
Next, solubilize the membrane suspension with 2%octal glucocide. Were 24 hours at 37 degrees Celsius. Protect the tube from light by wrapping it in aluminum foil.
Estimate the concentration of solubilized bacteria or dosin using the extinction coefficient listed in the text protocol just before reconstitution. Ultracentrifuge the solubilize bacterial opsin to get rid of lipids and un solubilize proteins. To prepare the liposomes in a glass speaker, weigh out 1.5 milligrams of cholesterol stored as a powder and add 400 microliters of diol phosphatidylcholine or DOPC dissolved in chloroform in a glass beaker, dry them under vacuum for at least one hour in a glass beaker just before manipulation.
The resulting DOPC to cholesterol molar ratio is 3.3 to one. After they have dried hydrate the lipids with one milliliter of hebes buffer, the suspension should appear turid. At this stage, heat the solution for 10 minutes at 37 degrees Celsius sonicate for 10 minutes at 40 watts with alternating 32nd pulse, 32nd pause cycles at room temperature, the suspension should appear clear.
At this stage, The most critical aspect of the procedure is obtaining a tight and mono dispersed suspension of liposomes. In order to do so, we take extreme care in setting up the extra ring device so that no leakage occurs. In addition, we clean the device very meticulously after each use.
To obtain a mono disperse population of liposomes, perform two cycles of extrusion for each cycle. Pass the liposome suspension through the filter at least 11 times. For the first cycle, use a membrane pore size of 200 nanometers, and for the second cycle, a membrane pore size of 100 nanometers.
At this point, dynamic light scattering measurements can be performed to check the homogeneity of the suspension. Shown here is a typical result of this step. Membrane proteins can be reconstituted in liposomes due to the solubilizing effect of detergents detergent.
Solubilized liposomes are incubated with the detergent solubilized membrane protein and formation of the protea. Liposomes is triggered by rapid elimination of the detergent with polystyrene beads. To incorporate protein, add 28 milligrams of tritton X 100 and incubate overnight at four degrees Celsius.
Add the detergent solubilized protein, including bacteria opsin Mex B and Mex A to the solubilized liposomes and incubate for 15 minutes at four degrees Celsius. Add previously activated polystyrene beads. Add a 30 to one bee to detergent ratio and incubate for five hours in the dark at room temperature.
Under gentle stirring, purify the protia liposomes protein and free substrates using a PD 10 desalting column equilibrated with he Hess buffer. To check that both the Mex B and the bacteria opsin have been reconstituted in liposomes, purify the protea liposome suspension on a discontinuous sucrose gradient. Gently settle the proteosome on a five layer gradient of sucrose.
Then ultra centrifuge for 17 hours at hundred thousand G following ultra ification, aggregated proteins are found at the bottom of the tube while non incorporated detergent solubilize proteins are found at the top of the tube. Protea, liposomes and liposomes are found at sucrose interfaces that correspond to their intrinsic density. Empty liposomes are recovered further up in the gradient than the prot liposomes.
Carefully collect the various gradient fractions before analyzing them with sodium dyl sulfate. Poly acrylamide gel electrophoresis using kumasi staining or western blotting. Perform fluorescence measurements at 25 degrees Celsius using a spectral fluorimeter, allowing for dual wavelength measurements.
Alternate the illumination periods to activate bacterial rodin characterized by excitation and emission wavelengths of 550 nanometers with measurements of purine fluorescence, which are characterized by an excitation wavelength of 455 nanometers and an emission wavelength of 509 nanometers for two seconds. Set the excitation and emission bandwidths to five nanometers. Perform the measurements in the presence of 50 nanomolar liny to prevent the formation of a reverse membrane potential.
Delta sigh shown here is a representative control obtained with the assay in the absence of Mex a, Mex B, verifying that the proton gradient generated by the bacterial rod opsin is reversible. After one cycle of acidification, Oreo liposomes are kept in the dark for 45 minutes in order for the bacteria opsin to stop pumping protons. After this recovery time activation of bacteria opsin is possible simply by illuminating the same suspension.
Again, an actual transport measurement shows that a negative control with protein free liposomes has a constant pH upon illumination as expected. However, protea liposomes containing bacteria or opsin in their membranes do pump protons. Thus, the pH inside the liposomes decreases and a stable gradient is built.
Gray triangles represent the pH inside of protea liposomes containing bacteria or opsin and mex B in the presence of hoax. 3, 3, 3, 4, 2 dye. While the red diamonds represent the pH in the absence of the dye, A substrate independent activity is observed where a proton gradient is only partially dissipated by Mex B counter transport, which is attributed to a basal activity of Mex B.In order to test the effect of mex A on the activity of Mex b Mex A was added to the reconstitution first.
In the absence of any substrate, again, the proton gradient generated by the bacterial opsin is only partially dissipated. The actual transport measurement is realized on the very same sample and the proton is rein initialized, and the substrate is added in the suspension in order to reach its binding site in the protein. Upon subsequent illumination, one can see that the proton gradient generated by the bacterial opsin is now totally dissipated by Mex AB as a consequence of substrate transport by the pump, It's important to keep in mind that exact condition necessary for adapting this protocol to another protein might vary from what has been described here.
Match care must be taken in optimizing the following parameters, quality of the purification, efficiency of the detergent, solubilizing step desorption of the detergent, lipid composition, and lipid to protein ratio. In addition, temperature and incubation time periods are prone to affect all these parameters. Our protocol opens the way towards a better understanding of the activity of the r and d flix pump mix B.Nevertheless, if it has been specifically sold for r and d transporter, this method could be adapted to any PMF activated FFL spam transporter.
