You know, chemistry is kind of a big field. I say that because I’ve been actively reading the chemical literature for over thirty years now, and I still keep running across topics that I never knew existed. One of these popped up the other day: racemic protein crystallography. Now there may be a few readers who are all “Oh yeah, sure, you’re just getting around to that?”, but for everyone else, think about that term for a minute. Prepare yourself for a weird excursion into chirality and crystallinity.
Protein crystallography, sure – great stuff, although known to be infested with voodoo and all that. But racemic? The idea of a racemic protein is a bit weird, because that means a 50/50 mix of enantiomers, and proteins are, of course, pretty much all made up of the naturally (L) amino acids that living cells use crank them out. To get an all (D) protein, you’d have to have a supply of all those unnatural enantiomeric amino acids (which you can buy, albeit not so cheaply). But you’re not going to be able to depend on any cells or ribosomes to make them for you, since we don’t have any mirror-image life handy, so chemical synthesis it is. And that’s doable, too (although the larger the protein chain, the trickier it gets).
But the next question you might have is why anyone would want to go to all the trouble. And there we get into some crystallography. If you look at all the basic ways that things can be arranged in three dimensions, with careful attention to what directions and how you’d have to move your basic units around to make them repeat, you are listing “space groups“. And here chemistry (and physics) impinge on pure mathematics, because good ol’ three-dimensional space has 230 different space groups: that’s all there are and there ain’t no more. There are several different ways to arrange them and more than several notations for them, as that last link will show you, but mathematically, that’s what we have to work with.
If you sit around your whole career crystallizing things, you will eventually encounter every single one of them, although (to be sure) there are definitely more and less common ones. But if you do nothing but crystallize proteins, you poor person you, then you will only encounter 65 of them at most. Those are the chiral space groups that don’t have any of the slightly fancier symmetries in them, such as inversion centers or mirror reflections, because chiral protein chains simply cannot be arranged in ways that will allow those things to happen.
And as it turns out, if you look at all the protein crystals known and bin them out into those 65 space groups, the P212121 group is pretty heavily over-represented, and a few others are right behind it. That link goes into what seems like the explanation: entropy. Those space groups allow for more degrees of freedom, which means that there are (in most cases) simply more opportunities for proteins to fall into that sort of pattern.
Now if you were to go to racemic proteins and crystallize those, you end up with 165 possible space groups, which means that crystallization should, in fact, be easier in general (since there are more ways for it to happen). And since protein crystallization (as it stands) can be a barren wasteland of heartbreak, this is good news. The statistics for globular proteins are that about 30% of them can be crystallized through brute-force experimentation, but only about two-thirds of those crystals, at best, give enough high-quality diffraction data to be useful.
But it really does look like the success rates for racemic crystallization are higher: greater than 85%, actually, which is just unheard of. An outstanding example is ubiquitin, a relatively small and very biologically important protein that is a major pain to crystallize. Its structure was determined in 1987, but as I understand it, almost everyone who studied its crystalline form since then did so by obtaining seed crystals from that original successful team (!) Synthesis of the enantiomeric protein made a dramatic difference: a standard screen of protein crystallization conditions on the racemate gave diffraction quality crystals in half the wells after an overnight run. Recently it was shown that this even applies to ubiquitin oligomers, which have been considered particularly intractable.
The racemic-protein idea was proposed in 1989 and first achieved in 1993, and it’s been gathering momentum ever since. The same paper that explained the current protein space group bias predicted, through the same considerations, which new space group(s) would become the most favored as more racemic proteins were studied and according to this recent review, they’re right on target (between 40 and 50 examples are known to date). The prediction was also made that membrane-bound proteins might be difficult, since they have fewer protein-protein contact points than soluble proteins tend to. Work in this area is still at an early stage, but so far it looks like you can get X-ray structures for such things, but that they might differ (on a macro subunit-assembly level) from what occurs in the real membranes.
This is all great if you can get past the brutal synthesis part, and that last review and this shorter recent overview go into that issue. What’s really made the difference in recent years is “native chemical ligation” chemistry on shorter (and unprotected) protein subunits. It’s still not something you undertake lightly, but it’s a lot more feasible than it used to be. And that’s a good thing: protein crystal data is extremely useful (although not quite the Word of God in all situations) and anything that allows us to get more of it, particularly in the tough cases, is welcome indeed.
Addendum: I can’t resist noting the first time I ever came across the idea of a fully enantiomeric protein, which was when I was about 14 years old and reading James Blish’s (rather odd) one-off short Star Trek novel, Spock Must Die. Blish (as always) couldn’t resist working his own interests into it, so you have oddities like Uhura being a devotee of James Joyce and Finnegan’s Wake in particular, which raised my eyebrows, Spock-like, even as a 14-year-old. At any rate, a key plot point is the emergency trial of a souped-up long range transporter beam that sends a copy of the person involved. Spock gets into the thing – mind you, they should have tried it on a box lunch of roast beef sandwiches or something first, but that’s the 1960s Star Trek – and the beam ends up unexpectedly reflecting back and producing an apparently identical Spock, to great consternation, not least because there’s some confusion about which one is the original. One of the two ends up barricading himself inside Dr. McCoy’s laboratories for reasons unknown, and when they discover that he’s the duplicate, one proof is that they find that he’s been in there synthesizing enantiomeric proteins and carbohydrates in order to survive. That reflection reversed him down to his molecular chirality, you see, so. . .