The overall goal of the following procedure is to obtain high quality crystals of a membrane protein for structured determination by X-ray crystalography. This is achieved by protein reconstitution into the membrane of a lipic cubic phase and incubation with a precipitant solution, which induces crystal nucleation and growth due to a vast size of crystallization space and a limited feedback from crystallization experiments. The lipic cubic phase fluorescence recovery after PHOTOBLEACHING or LCP Frap Prec crystallization assay was developed to select for the most promising crystallization conditions.
This assay reduces the amount of screening and increases chances of obtaining initial crystallization hits. Once initial hits are found, optimization trials are set up in order to improve crystal growth. Finally, optimized crystals are harvested and frozen in liquid nitrogen for data collection at a synchrotron source.
Results show the effective use of the LCP FRA assay for obtaining initial crystals of BE two two adrenergic receptor in LCP, of which further optimization led to diffraction quality crystals. This technique takes advantage of a native-like membrane environment provided by the repeated cubic phase, and typically results in crystals with low solving content and better ordering as compared to crystalization and detergent solutions. Generally, individuals new to this method will struggle because of the high viscosity at a Haitian properties of lipid mass phases making this materials difficult to handle.
However, when the right tools are used and technique is mastered, this procedure is no more difficult than any other protein crystallization technique and is amenable to automation and high throughput. So let's get started. Purify membrane protein of interest in a detergent solution and concentrate the protein detergent complexes to approximately 10 to 20 milligrams per milliliter.
Transfer about 25 milligrams of A LCP host lipid or a lipid mixture into a plastic tube, and incubate at 40 degrees Celsius for a few minutes until the lipid melts. Next, attach a syringe coupler to a 100 microliter gast tight syringe. Load the syringe with the molten lipid using an adjustable volume pipette.
Record the volume of the lipid in the syringe load another 100 microliter syringe with enough protein solution to result in a protein solution to lipid ratio of two to three volume by volume. Connect both syringes together through the syringe coupler. Push the syringe plungers alternatively to move the lipid and protein through the inner needle of the coupler back and forth until the lipid meso phase becomes homogeneous.
LCP forms spontaneously upon mechanical mixing and the protein becomes reconstituted in the lipid bilayer of LCP. Formation of LCP can be verified by its transparent and gel-like consistency, and by the absence of birefringence when viewed under a microscope, equipped with cross polarizers or if possible by using small angle x-ray diffraction. To begin LCP frap assays, label the protein with a fluorescent diet at a protein to die ratio of about 100 to one as described in the written protocol.
Remove the unreactive dye and concentrate the protein to approximately one milligram per milliliter. Next, reconstitute the labeled protein in LCP in the same manner as done for the unlabeled protein. Set up assay plates.
Using LCP wrap screening solutions. By first transferring the protein laden LCP into a 10 microliter gas tight syringe attached to a repetitive syringe dispenser, then attach a short removable needle to the 10 microliter syringe. Dispense 200 nanoliter boluses of LCP on the surface of four adjacent wells, forming a two by two square overlay each of the LCP boluses with one microliter of the corresponding LCP FRA screening solution.
Once the screening solution has been dispensed, cap the four loaded wells with an 18 millimeter square glass cover slip. Apply a gentle pressure on the cover slip to seal the wells. Repeat this process for the next set of four wells until the whole plate is filled.
Once filled, cover the plate with foil. Incubate the plates at 20 degrees Celsius for at least 12 hours. To achieve an equilibrium state place one of the plates on the LCP Frap station and focus on the first well using a 10 times objective.
The frap data acquisition is performed automatically using an image pro script in which the user inputs all acquisition parameters first and then presses the start button. A typical acquisition sequence starts with recording five pre bleached images, then triggers the bleaching laser synchronized with streaming a fast recovery sequence of about 200 images at the fastest possible rate, followed by taking a slow recovery sequence of 50 images with intervals of one to 20 seconds between images depending on the protein diffusion rate. After data collection is finished, integrate the intensity inside the bleach spot and for reference spots placed outside of the laser bleached area in all frames correct.
The integrated intensity curve for deviations caused by fluctuations of light and bleaching during the data acquisition normalize the corrected intensity to make the pre bleached intensity equal to one and the initial bleached intensity equal to zero, fit the curve of the normalized intensity versus time as described in the written protocol to obtain the mobile fraction and the diffusion coefficient. Repeat this procedure for the next, well compare the mobile fractions and diffusion coefficients obtained for the different screening conditions. Design nu crystallization screens based on the components that facilitated protein diffusion and excluding conditions for which protein diffusion was not observed to set up LCP crystallization trials.
Reconstitute the protein in LCP as before, then set up crystallization plates as for LCP frap, but with crystallization screening solutions. Alternatively, crystallization trials can be set up robotically once crystallization plates have been prepared, incubate them at a constant temperature, periodically checking for crystal formation and growth. Once the protein crystals have grown, place a plate with the crystals under a stereo microscope with variable zoom equipped with a linear rotating polarizer and analyzer.
Focus on the well of interest using a low power zoom so that the whole well is placed within the field of view. Score the cover slip glass in four strokes, making a square inside the well boundaries using a sharp corner of a ceramic capillary cutting stone. Press around the scored perimeter with strong sharp point tweezers to propagate the scratches through the thickness of the cover.
Slip glass punch two small holes at opposite corners of the scored square. Inject a few microliters of precipitant solution through one of the holes to reduce dehydration during the subsequent steps. Using an angled sharp needle probe, break up the glass along one or two sides to free the cutout square.
Carefully lift up the glass square watching for the cubic phase bolus. Add an extra few microliters of precipitant solution supplemented with a cryoprotectant if necessary on top of the exposed cubic phase bolus in the well increase magnification of the microscope and focus on a crystal. Adjust the angle between the polarizer and the analyzer to increase the contrast between the frt crystal and the background while keeping enough light to see the harvesting loop.
Then remove excess lipid and harvest the crystal directly from the LCP by scooping it into a mighty gen micro mount that has been selected to have a diameter matching the crystal size flash. Freeze the microm mount with the harvested crystal in liquid nitrogen and ship it to a synchrotron source beamline for x-ray data collection engineered beta two adrenergic receptor was labeled with S SI three NHS ester and used in LCP Frep prec crystallization assays in automatic high throughput mode in which each sample of a 96 well plate is bleached sequentially and fluorescence recovery is measured after a 30 minute incubation. The obtained fluorescence recoveries, which represent the mobile fraction in each sample are plotted for all 96 samples.
The screening solutions contain 0.1 molar tris pH eight 30%volume by volume PEG 400, combined with 48 different salts at two different concentrations. Fluorescence recovery profiles for several representative conditions are shown. Solid line curves represent fits of the normalized intensity versus time fast recovery of less than 10%in the sample containing sodium chloride is due to fluorescently labeled lipids.
Co purified with the protein initial crystal hits of beta two adrenergic receptor in complex with olol were obtained by a coarse grid screening around the most promising conditions identified by LCP Frap containing sodium sulfate and sodium formate. Further optimization of precipitant conditions yield a diffraction quality crystals. Here, the images of crystals grown in the presence of sodium sulfate and potassium formate are taken in the brightfield mode.
The same crystals can also be viewed using cross polarizers. Once mastered reconstitution protein in lipid cubic phases can be co completed within 10 to 15 minutes, and a manual setup of a 96 well crystallization plate within one hour L three B wrap assay in high mode requires about two hours per 96 well plate, and the H crystal takes approximately 10 to 20 minutes per well. The procedures shown in this video provide a basic guide for approaching music crystallization trials.
However, as in any crystallization experiment, an extensive screen and optimization are required, and a successful outcome is not necessarily guaranteed.
