The Magic Angle
Occasionally coherent ramblings on anything that strikes my fancy....
Friday, September 03, 2004
Adventures in Babysitting (Or, Watch Michael Try to Grow Protein Crystals)
Most of us probably have somewhat fond memories of growing simple salt crystals when younger, either in school or out of the classroom. It's fun, it's easy, and it's actually kind of pretty after a fashion. For those of us who went on to take more science classes at the university level, you may have fond (OK, probably not-so-fond) memories of trying to crystallize a compound you synthesized in a laboratory course. Crystallization is an important step for many investigations of chemical structure and function for at least two reasons - it is generally a sign that what you have is reasonably pure (although not necessarily what you're looking for), and is also suitable for analytical methods where a solid is required. This, of course, is what is needed for x-ray crystallography. This method depends upon the fact that x-rays will diffract in a comprehensible way from a regular, periodic arrangement of atoms and/or molecules in a crystal. The sorts of salts we used to play with as children and organic compounds that we may have played with as young adults are not that typically large - perhaps equivalent to a few hundred daltons (where one dalton is equivalent to the mass of a single hydrogen). They are typically pretty anhydrous (one can heat them up, driving away any stray water molecules, without doing much damage - although heating it up too much will melt the salt, and that's a different story) and, for the most part, quick to prepare.
Proteins, however, are a different story. And here things get interesting. Protein crystals can have lots of associated waters. It's been found that some protein crystals are, in fact, mostly water that's bound to the protein in one way or another. Some protein crystals can take up to a month to grow properly. As some of you are probably saying to yourselves right now, proteins are generally far larger than a few hundred daltons - tens to hundreds of thousands of daltons, particularly when one is talking about large respiratory complexes and other such monsters of biochemistry. Some proteins are notoriously difficult to crystallize - membrane proteins, which reside in a greasy envelope of lipids or are associated to one via a greasy anchor at one end of the protein, are one of the continuing challenges for protein crystallographers. Instead of trying to line up in three dimensions small, cute, cuddly atoms and molecules, one is attempting to line up large, bulky, recalcitrant proteins into crystals on the scale of a few hundred micrometers in each direction ideally.
As to how to do this....there is a curious blend of physical chemistry, one's intuition, and black magic. (OK, I'm kidding about the black magic. But if you happen to have any goats available for moonlight sacrifices.....) The idea is that one needs to precipitate the protein out of solution slowly and in an orderly fashion. Salts are often added to the buffer so as to take up water molecules in a solvation shell which would otherwise be surrounding the protein, as well as to affect the surface residues of the protein. Organic molecules such as polyethylene glycol are also added, which draw away water from the proteins in a similar fashion. Other chemicals may be added as needed - for example, dithiothreitol or 2-mercaptoethanol to prevent formation of disulfide bonds, or perhaps if one is working with an enzyme, the enzyme's preferred substrate so as to see where the substrate binds relative to the active site. One can also play with temperature, the amount of protein, and the geometry of the crystallization process (do you have your protein hanging above a reservoir of these compounds, sitting down surrounded by these compounds, or being injected into a microfluidics device?).
Even after all of this, you may still not end up with a protein crystal. Or, you may not end up with a large enough protein crystal - one might have a batch of so-called "microcrystals," so-called since they're best measured not in hundreds of micrometers but far fewer micrometers. What might look like messy precipitate might be nanocrystals, but it could just be unordered protein precipitate after all. My lab's specialty - solid state NMR - is able to deal with such cases, where crystals don't form large enough crystals for x-ray diffraction studies or fails to form crystals at all.
However, to my astonishment, I managed to grow a very few crystals back in the spring of x-ray diffraction size and so I've been following up on that as well as more expected pursuits. You see, there are some interesting questions that arise from thinking about the currently available crystal structures for the system I work with when considering temperature effects and under what conditions one finds my protein (to wit, crystals grown in a refrigerator don't necessarily correlate to an organism which does not grow at such temperatures). So, I figured, why not? However, as was once related to me by a protein crystallographer, "Sometimes it's just a matter of setting enough trays and wells before you start getting crystals." So, as to what I actually do when attempting to grow crystals? Let me show you.
First, I take one (or two, or three, or however many I need) crystallization tray(s) (we use the well known standard from Hampton Research, their VDX Plate), and apply a small bit of vacuum grease to each and every circular well ring. This makes sure that when I apply the cover slide (which will come later), I have a reliable seal and will not have contamination from the outside. This is done by gently heating some vacuum grease until it melts and dipping a small glass flask upside down into the grease, and then carefully applying the greased rim of the flask to the well ring. When I first started doing this, it would take almost an hour to do this for an entire tray (24 wells) - I was very concerned about dripping grease into the wells and, in general, making a mess of the entire process. Now it takes ten minutes on a good day. I'll usually make sure that my precipitation/crystallization solutions are prepared and and ready to be added, either directly or after a small bit of mixing if I'm using a high concentration stock solution of polyethylene glycol. The protein solution is in its appropriate buffer and at a high enough concentration, and has been mixed with the substrate if I'm doing such a trial. Now, I use pretty high concentrations of protein at times (over 200 milligrams per milliliter on occasion), so I often have to use centrifugal concentration methods - like these from Fisher - to get my protein to the desired protein concentration. If I'm cocrystallizing my protein with its substrate, I'll typically do this before concentrating - this way I can monitor the binding with UV/Vis spectra and then be sure that my substrate is in there before spending all that time concentrating my protein to a thin layer of protein sludge (well, not really sludge per se, just rather viscous solution). I then take some small plastic microscope cover slips, add a small bit of protein sludge, add a small bit of precipitation/crystallization solution, and then place the slide over a filled well. This is one of those places beginners can botch things up - if you take too long in flipping the slide over, it can run and you get something smeared out over more area than a neat small drop's worth. You then gently press down and end up with a sealed well, protein hanging above your solution. You do this 12, 24, 48, 72, ..... times and you wait. You need to check daily under a microscope (just a regular light microscope, nothing fancy is really needed for just monitoring growth or lack thereof of your protein crystals, and we have a digital camera to take pictures of anything interesting looking) and wait. Did I mention the waiting? This is the babysitting, and what usually takes the longest. You can make up your solutions, prepare your tray with the grease seals, concentrate your protein, and mix everything up in a day - the waiting is what really takes the time. One day of work - a week or longer (far, far longer in some cases) of watching and waiting.
I've had middling to fair success thus far - one of the proteins I worked with grew crystals pretty easily (then again, the conditions were already known) and the other one, my main interest, is coming along reasonably well. I seem to be able to get microcrystalline material without too much difficulty, and am playing around with conditions to see if I can get larger crystals. I'm also going to try such interesting techniques as microseeding - taking a small microcrystal and dropping it into a freshly set well to act as a growth initiator for a larger crystal - and magnetic field crystallization - where one basically sets up a magnetic field surrounding your trial in order to grow larger crystals. (As for an actual explanation of how magnetic fields affect protein crystallization, your guess is as good as mine - there are lots of ideas, but nothing firm or even reliable. One can wave one's hands about, but in the end.....remember the comment about black magic before?) Something that I need to be careful about is how conditions for crystal growth may not be optimal for doing NMR - too much salt in microcrystals or precipitate can cause unwanted heated during an NMR experiment, considering that we're pumping in high power radiofrequency waves; too much precipitant (polyethylene glycol) might cause unwanted signals in the resulting spectra of the protein.
Ultimately, though, it's well worth the time - if I can devise better ways to precipitate out protein (forming large, x-ray grade crystals or not), the easier my adventures in preparing future NMR samples should be, as well as the tantalizing idea of having comparative crystallographic data to work with when trying to synthesize what is already known with my original research.
Sites To Look At:
Crystallography 101 - An introduction to x-ray crystallography at Lawrence Livermore.
Crystals for Fun and Education - Hampton Research has some fun and cool crystallization projects for kids (and those who are still kids at heart).
posted by Michael @ 11:35 PM
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