Hello, my team study type of protein last, span across the cellular membrane and we are looking at the relationship to the medicine and biology. Specifically, we are looking at one particular protein, which would require ATP to drive its function and then, is part, the name is called ATP-binding cassette transporter. In particular, we are looking at their fundamental structure-function relationship and then, use that to understand a lot of chronic disease, especially cardiovascular disease.
Due to the complexity of purifying the membrane proteins and their stability at native temperatures, obtaining 3D protein crystals is the main challenge in the field and it also limits the drug-discovery applications. Our team has established methods in cryo-EM and X-ray crystallography for the structural studies of membrane transporter proteins. And previously, our lab also solved the crystal structure of the first human ABCG5, G8 obligate heterodimer in both APO as well as cholesterol-bound states.
Our results can provide a breakthrough in small-molecule drug discovery or drug repurposing. Obtaining 3D protein crystals, can also enable soaking experiments for the cocrystallization of protein and ligand complexes. Our laboratory aims to determine the molecular basis of lipid recognition and translocation by ATP-dependent lipid transporter proteins.
And we also aim to establish new methods in drug development for studying the molecular dynamics and structural aspects of ABC transporters. Begin by chemically modifying the pooled protein fractions through reductive methylation. To do so, take the purified protein and add 20 millimolar dimethylamine borane, followed by 40 millimolar formaldehyde to it.
Incubate the mixture in an oscillatory shaker for two hours at four degrees Celsius. Then, add an additional 10 millimolar dimethylamine borane to the mixture. Next, incubate the mixture overnight for 12 to 18 hours at four degrees Celsius.
Quench the reaction by adding 100 millimolar tris chloride, having a pH of 7.5. Use the appropriate wash buffer and elution buffer for loading the methylated protein onto a nickel NTA column. First, wash the two milliliter nickel NTA column with 10 column volumes of wash buffer.
And then, elute the protein with the elution buffer. Following Elution, pass the protein eluent through a PD-10 desalting column, pre-equilibrated with the appropriate buffer. Add cholesterol prepared in isopropanol or ethanol to the desalted protein.
And incubate the mixture overnight at four degrees Celsius. The next morning, ultracentrifuge the mixture at 150, 000 G for 10 minutes at four degrees Celsius. Add the collected supernatant to a centrifugal concentrator with a 100 kilodalton cutoff and concentrate the supernatant to a final concentration of 30 to 50 milligrams per milliliter.
Once more, centrifuge the concentrated protein, using a high-speed benchtop refrigerated centrifuge and remove the pellet. Place the collected supernatant on the ice at four degrees Celsius before proceeding to set the crystallization. Alkylated proteins stored at four degrees Celsius were analyzed by analytical gel filtration chromatography over the course of a month.
And it showed a slight protein loss after a week. Begin by preparing a 10%B-cell stock solution, containing the lipids and detergent. To do so, take the pre-dried lipid, which is a mix of five mole percent cholesterol and 95 mole percent DMPC and add the CHAPSO detergent solution in deionized water to it.
Using a water bath sonicator, resuspend the lipid-detergent mixture by sonicating it in ice-chilled water under continuous power until the solution is clear. Then, with the aid of a 0.2 micron centrifugal filter, remove any undissolved components. Next, working on the ice, combine the resultant B-cell stock solution with the pre-prepared protein sample at a one to four volumetric ratio.
Incubate the protein B-cell mixture on ice for 30 minutes. For crystallization in a 48-well plate, mix 0.5 or one microliter of the protein B-cell mixture with an equal volume of crystallization reservoir solution. Then, set up crystallization in a hanging drop vapor diffusion mode, using the 48-well plate and incubate the crystallization setup at 20 degrees Celsius.
On the following day, check the crystallization trays to ensure the cover glass is sealed properly. Check on the crystal growth at least once daily, using a tabletop stereo microscope equipped with a polarizer. Lastly, soak the protein crystals in 0.2 molar sodium malonate and flash-freeze them in 50 or 100 micrometer cryo loops, using liquid nitrogen.
Mature crystals that were suitable for data collection usually appeared within one to two weeks and generally, had the dimensions of 50 microns by 100 microns by two microns.
