脂質中間相を用いた構造決定のための膜タンパク質の結晶化

Hello and welcome to the Membrane Structural and Functional Biology Group. My name is Martin Caffery. The focus of our research is the biological membrane, the schematic of which is shown on the first slide, the membrane which surrounds the cell and subcellular organelles when present is a molecularly thin structure, just two lipid molecules across and studded with proteins.

We are interested in structure and functions applied to both lipids and proteins. However, for purposes of this JO series, I will limit my focus to membrane proteins. We seek to understand how these membrane proteins function at a molecular level for two reasons.

Firstly, there is the intellectual satisfaction in knowing how something works. Secondly, by knowing how it works, just like your bicycle or car, there is the prospect of fixing it if it malfunctions. Drug design is an obvious outcome of this type of work.

The approach we take to figuring how a membrane protein works at a molecular level is to determine its crystallographic structure. This involves establishing the location in three dimensional space of all atoms, or at least all non hydrogen atoms that make up the protein. The method we use for this purpose is called macromolecular X-ray crystallography MX for short.

Here we see an example of a membrane protein whose structure was determined using MX at a fraction quality crystal of the protein is required to do mx. As you can imagine, there are many steps involved in structured determination using macromolecular crystallography as illustrated.Here. Typically these include identifying a membrane protein target and then producing purifying and crystallizing it.

Diffraction measurements are performed on the crystal using a home or a synchrotron x-ray source. The diffraction data are processed, yielding an electron density map, which is then fitted with a molecular model. The model when refined can be used to explore the mechanism of action of the protein and for structure-based drug design.

The focus of this JO of article is to show you how we produce a fraction quality crystals of membrane proteins using lipid meso phases by the so-called in meso method. A recent review of the method and its scope is available in reference one. The step-by-step protocol we will follow here is described in reference to a flow chart summarizing the steps involved in and time required for setting up an end meso membrane protein crystallization trial is shown here.

This video covers those steps enclosed by dashed red lines. The items you will need to do in meso crystallization in the manual mode are shown here. They include a concentrated solution of the purified membrane protein lipid for creating the hosting mease, usually mono ole to make the lipid more obvious.

In this video, it has been doped with red dye precipitant solutions, purified water pipe petting devices and disposable tips and assortment of gast tight Hamilton syringes, some with removable needles, a narrow bore coupler used to mix the protein solution with the lipid to produce the mease. A repeating dispenser from Hamilton glass microscope slides and cover slips. Ideally siloized perforated double stick spacer tape for well creation tweezers, chipped ice tissues, a calculator and most important of all your lab notebook.

The following procedure is used to prepare a glass sandwich crystallization plate. For loading, place a siloized microscope slide on the bench. Remove the protective paper cover from one surface of a strip of perforated double stick spacer tape.

Place the tape sticky side down in contact with the surface of the slide pressure. Seal the tape to the slide. Using a brayer or roller aluminum foil can be placed between the tape and the roller to protect the base of the wells.

Remove the second paper cover from the tape to expose its upper sticky surface. This procedure generates a glass plate that is ready for loading and subsequent ceiling as soon as loading is complete place. Individual siloized cover slips in close proximity to the plate for fast and efficient ceiling of wells.

Place the lipid in a temperature controlled block at 45 degrees Celsius for three minutes to render it molten. Note that the lipid used is typically mono land. In this video, the lipid has a red dye added for visibility while the lipid is melting.

Prepare two 100 microliter gas tight Hamilton syringes for use In the lipid protein mixing step, remove the Teflon fur oil from the syringe that will contain the protein solution. Place the syringe that will hold the lipid next to it. In this case, the fal is left in place.

Place the narrow bore coupler between the two syringes and then connect the coupler to the lipid syringe finger. Tighten the coupler to the syringe, but do not overtighten. Remove the plunger from the barrel of the lipid syringe.

Make sure the lipid is molten. Set the pipet to 30 microliters and slowly take up 30 microliters of molten lipid cap. The lipid vial.

The lipid should be stored under nitrogen or argon at minus 80 degrees Celsius. Slowly deliver as much of the molten lipid as he can into the open end of the syringe. Taking care not to introduce air gaps.

Place the plunger into the barrel in contact with the molten leopard and slowly advance the plunger up the barrel with the syringe held vertically. As shown, the air bubble trapped inadvertently rises in the process and is released. We now have a plug of molten lipid in the barrel whose volume you must determine accurately.

This can be done by reading the markings on the syringe barrel. In this case, the volume is 23 microliters, which is recorded in the lab notebook. Slide the fur onto the needle extending from the coupler.

Use the plunger to force the molten lipid up the barrel and slowly and gently into the needle at the core of the coupler. If the needle is slightly overfilled, the lipid will be seen beating out at its open end. By backing up the plunger slightly, the excess lipid can be withdrawn into the coupler until it is just flush with the tip of the coupler needle.

In this case, the syringe coupler unit is fully loaded and ready to be combined with the protein syringe. The solution should be spun at 14, 000 G for 10 min, five to 10 minutes at four degrees Celsius to remove large aggregates. Before setting up crystalization trials, take the protein solution from the ice bucket and equilibrate it at room temperature.

Calculate the volume of protein, so solution to use to form the cubic phase at or close to full hydration for olean a 20 degrees Celsius. Full hydration with water occurs at close to 40%by weight water as in the phase diagram. Recall that we have loaded the lipid syringe with 23 of mono at a density of 0.94 milligrams per microliter.

This corresponds to 21.6 milligrams of lipid, thus 21.6 times four divided by six or 14.4 milligrams or 14.4 microliters of protein solution is needed with a 25 or 50 microliter Hamilton syringe take up the required volume of protein solution, transfer the solution onto the Teflon tip of the plunger in the barrel of the protein solution. Taking care to avoid air bubbles carefully withdraw the needle and measure the volume of protein solution in the syringe. It should match the value delivered in the previous step.

In preparation for combining with the lipid loaded syringe. Carefully inch the protein solution up the barrel to end up flush with its open end. This can be observed by looking down through the barrel termination end.

The lipin protein solution, loaded syringes are now ready to be combined in preparation for Mex to do this, screw the protein syringe into the open end of the coupler attached to the lipid syringe. Having combined two syringes by way of the coupler. A continuous volume of liquid should exist from lipid in one syringe through the coupler to protein solution in the other syringe.

In preparation for mixing, the assembled unit is held in one with one syringe in the right hand and the other in the left. Torque is applied to both barrels through to the coupler, using the fingers and thumb of both hands to ensure that the seal against the furrows in the coupler remains intact over tightening. The assembled mixing device at this stage will damage the ALS and cause leaking under tightening will also lead to leaking.

It is a matter of experience with the inevitable trial and error and occasional loss of sample. It's worthwhile therefore practicing with lipid and water or buffer solution to get a feel for the system before launching into using valuable protein solution to affect mixing, advance the plunger on the protein side of the assembled mixing unit to its limit with the thumb or index finger driving the protein solution out of the protein syringe through the coupler and into the lipid syringe. If the unit has been sealed effectively, this action will cause an equal and opposite movement of the plunger on the lipid side.

The plunger on the lipid side is now used to drive the contents of the lipid syringe back through the coler and into the protein syringe. This process is repeated many times. Occasionally a hundred passages or more are required to produce a homogenous misa phase.

At the start of mixing, movement of material back and forth through the coupler can be uneven and at times extra force is needed to to effect mixing. This is to be expected. Initial mixing is usually accompanied by the development of a non-uniform cloudiness in the sample and again is expected.

As homogenization progresses. The texture as sensed by the force needed to move the plungers back and forth becomes more uniform and characteristic of the viscus cubic phase, as does the visual appearance of the emerging cubic phase. If conditions are appropriate and the cubic phase forms, the dispersion should appear optically transparent in the syringe barrel.

Thus, the markings on the syringe barrel should be clearly legible through the meso phase in the barrel. In the case of colored proteins, the meso phase will have the color of the protein, but will be optically transparent. Very slight cooling of the sample during mixing by placing the syringe mixer for a short time on ice can accelerate homogenization and the achievement of transparency.

However, it is important not to overco the sample care should be taken to avoid extremely vigorous mixing as this can cause sample temperature to rise due to frictional heating. At this juncture, the cubic misa phase has been formed and the protein is reconstituted into the lipid blay. The misa phase is now ready for dispensing into individual wells of the crystallization plates.

First, however, the meso phase must be transferred to a 10 microliter Hamilton syringe mounted on a repeating dispenser. Begin this process by coupling the syringe to the dispenser thin. Load the syringe with the cubic mease.

Remove the needle and the plunger from the syringe. Leave the Teflon fal in place. Remove the retaining nut from the dispenser with the aid of a small coin.

With the ratchet arm fully withdrawn. Insert the syringe without its plunger and needle through the holding ring of the dispenser. Replace the retaining knot gasket side facing the syringe and screw it in tightly on the open end of the syringe.

Care should be taken to ensure the syringe is properly centered in the holding ring and that the barrel is aligned parallel to the ratchet arm. Both can be judged by I.These two requirements are important because if the plunger does not run free and through pressure will build up in the source syringe during loading and leakage can occur. Pass the plunger through the gripping ring with the gripper nut, unscrewed a few turns and guide the plunger into the open end of the syringe.

Press the ratchet arm fully move the plunger into the barrel and check that it travels freely and through in the barrel. If the screw and the guide bar was loosened, re-tighten it and recheck that the plunger moves freely. I the dispensing syringe is now ready for loading.

Move the plunger on one side of the assembled mixing unit to the zero microliter graduation mark. To transfer the meso phase to the other syringe and the coupler. Disconnect the empty syringe with its UL in place from the mixing unit and immediately connect the loaded syringe with the coupler attached to the threaded termination of the 10 microliter dispensing syringe.

The degree of tightness with which coupling is done is critical. As already noted, loaded the dispensing syringe by depressing the plunger of the 100 microliter syringe so that the meso phase transfers through the coupler. If coupling is tight and the plunger in the dispensing syringe runs free, then the plunger should begin to move in the direction of motion of the loading plunger as the dispensing syringe fills.

Disconnect the loading syringe with the coupler attached from the dispensing syringe. Secure a short, flat tipped needle to the steel termination of the dispensing syringe and carefully tighten it in place. Clamp the plunger to the ratchet arm by tightening the knot in the gripping ring.

Care should be taken not to overtighten the knot. As this can score and deform the plunger rendering it unusable. It is important that the travel range provided to the ratchet arm in the clamped condition be limited to no more than an inch.

As shown beyond this, the plunger will have a tendency to buckle and delivery can fail. L depress the ratchet drum several times to advance the plunger in the barrel, thereby filling the void volume of the needle and loading the needle. This step should be repeated up to 10 times until a continuous string of the mesa phase emerges from the tip of the needle.

The needle is now ready for use in setting up crystallization trials, which should commence immediately. It is not advisable to keep the protein loaded cubic phase for too long before setting up the crystallization trials. As some proteins are unstable in the cubic phase without added precipitant, place a multi-well crystallization plate and cover slip on a surface, raised a few inches above the bench for ease of loading.

Optimal contrast and enhanced visibility are achieved when the surface is slightly dark. Homogenize the precipitant solutions and uncapped the vials. Set the precipitant dispensing proppe to one microliter with the dispensing syringe held vertically in one hand, use the free hand to position the needle tip in the center and directly above the base of well.

Number one. Press the button on the dispen on the repeating dispenser to expel a bolus of mease onto the glass surface. The bolus volume is 200 nanoliters.

When the standard repeating dispenser is used with a 10 microliter syringe, the tip of the needle should be no more than a few hundred micrometers above the base of the well To ensure proper delivery, reproducible delivery is easily achieved. It just takes a little practice. After the four adjacent wells are loaded with mease, place one microliter of precipitant solution on top of each mease bolus.

Using a two microliter per patent and standard disposable tips as quickly as possible, place a cover slip squarely over the filled wells to cover them uniformly to effect a watertight seal. Use a spatula to apply pressure to the cover slip where it makes contact with the exposed stick sticky surface of the spacer tape. The mease and precipitant dispensing process can be repeated until all wells on the plate are loaded and sealed.

The plate is now ready for inspection and for incubation. Label the plate clearly for tracking purposes. Light sensitive proteins are usually handled by wrapping the plates in aluminum foil before placing them in the incubation chamber.

These can be removed and examined under the microscope in subdued or appropriately colored light. Place the plates in a temperature controlled chamber usually at 20 degrees Celsius or thereabouts On a regular schedule. Inspect the walls for crystal growth using a polarized light microscope with a 10 or 20 fold met.

Objective, the schedule used in the author's lab is as follows, day 0 1 2 3 5 7 14 21 30 post set. Carefully inspect the mease bolus. Adjusting the depth of focus within the 140 micron thick sample.

Examination should be done both in normal light and between cross crossed polarizers, colorless membrane protein crystals growing in the cubic phase when viewed with normal light. Look like this. Colorless membrane protein crystals growing in meso when viewed with polarized light look like this or this naturally colored membrane proteins growing in meso when viewed with normal light look like this or this.

The next steps in the overall process of structured determination are to harvest and cryo cool the crystals, and to record and process x-ray diffraction from them. These topics are covered in separate JoVE articles in this series.