Now that I have managed to purify the full-length huntingtin protein with 3 different polyQ lengths (Q23, Q46 and Q78), I can begin the more exciting experiments where I can try to understand how the protein molecule is folded and what its structure is.
One method of doing this is called electron microscopy (EM). This involves taking a pure solution of the protein (made in the previous series of experiments) and putting it on a very small grid. By hitting the grid with an intense electron beam and using detectors which can work out how the electrons are scattered by the sample, we can image the individual protein molecules. The protein is very small, approximately 100,000th of a mm. We can take pictures of the protein molecules sitting in the holes of the grid using the microscope and from this complete a 3D reconstruction of what the protein molecule looks like in 3D space. We hope that this will reveal how the polyQ length might affect the protein fold and structure which could give rise to a change in function.
The grids have to be imaged whilst they are cooled with liquid nitrogen so the protein particles have to sit in a thin layer of ice in the grid. The ice layer needs to be approximately as thin as the protein molecules which are being imaged so that they sit in a single layer, not overlapping in the depths in the ice. They also need to be spread out enough that they don’t overlap laterally. This process is further complicated by particles binding to the grid surface itself and not sitting in the holes, so calculating a good concentration of protein to use is not trivial. The ice also needs to be formed very quickly, we can’t just pop the grids in the freezer, we have to plunge them into liquid ethane in order to form a vitreous ice layer. All in all, it is somewhat complicated and difficult to make these grids!
I have been working hard with Susan Lea all week and we have tried and tested a number of conditions to get the smooth thin ice with a good spread of huntingtin particles. We are almost there I think! My summary of this work is on Zenodo. More to come soon.