We're getting closer to real-time X-ray structures of protein function, and I think I speak for a lot of chemists and biologists when I say that this has been a longstanding dream. X-ray structures, when they work well, can give you atomic-level structural data, but they've been limited to static time scales. In the old, old days, structures of small molecules were a lot of work, and structure of a protein took years of hard labor and was obvious Nobel Prize material. As time went on, brighter X-ray sources and much better detectors sped things up (since a lot of the X-rays deflected from a large compound are of very low intensity), and computing power came along to crunch through the piles of data thus generated. These days, x-ray structures are generated for systems of huge complexity and importance. Working at that level is no stroll through the garden, but more tractable protein structures are generated almost routinely (although growing good protein crystals is still something of a dark art, and is accomplished through what can accurately be called enlightened brute force).
But even with synchrotron X-ray sources blasting your crystals, you're still getting a static picture. And proteins are not static objects; the whole point of them is how they move (and for enzymes, how they get other molecules to move in their active sites). I've heard Barry Sharpless quoted to the effect that understanding an enzyme by studying its X-ray structures is like trying to get to know a person by visiting their corpse. I haven't heard him say that (although it sounds like him!), but whoever said it was correct.
Comes now this paper in PNAS, a multinational effort with the latest on the attempts to change that situation. The team is looking at photoactive yellow protein (PYP), a blue-light receptor protein from a purple sulfur bacterium. Those guys vigorously swim away from blue light, which they find harmful, and this seems to be the receptor that alerts them to its presence. And the inner workings of the protein are known, to some extent. There's a p-courmaric acid in there, bound to a Cys residue, and when blue light hits it, the double bond switches from trans to cis. The resulting conformational change is the signaling event.
But while knowing things at that level is fine (and took no small amount of work), there are still a lot of questions left unanswered. The actual isomerization is a single-photon event and happens in a picosecond or two. But the protein changes that happen after that, well, those are a mess. A lot of work has gone into trying to unravel what moves where, and when, and how that translates into a cellular signal. And although this is a mere purple sulfur bacterium (What's so mere? They've been on this planet a lot longer than we have), these questions are exactly the ones that get asked about protein conformational signaling all through living systems. The rods and cones in your eyes are doing something very similar as you read this blog post, as are the neurotransmitter receptors in your optic nerves, and so on.
This technique, variations of which have been coming on for some years now, uses multiple wavelengths of X-rays simultaneously, and scans them across large protein crystals. Adjusting the timing of the X-ray pulse compared to the light pulse that sets off the protein motion gives you time-resolved spectra - that is, if you have extremely good equipment, world-class technique, and vast amounts of patience. (For one thing, this has to be done over and over again from many different angles).
And here's what's happening: first off, the cis structure is quite weird. The carbonyl is 90 degrees out of the plane, making (among other things) a very transient hydrogen bond with a backbone nitrogen. Several dihedral angles have to be distorted to accommodate this, and it's a testament to the weirdness of protein active sites that it exists at all. It then twangs back to a planar conformation, but at the cost of breaking another hydrogen bond back at the phenolate end of things. That leaves another kind of strain in the system, which is relieved by a shift to yet another intermediate structure through a dihedral rotation, and that one in turn goes through a truly messy transition to a blue-shifted intermediate. That involves four hydrogen bonds and a 180-degree rotation in a dihedral angle, and seems to be the weak link in the whole process - about half the transitions fail and flop back to the ground state at that point. That also lets a crucial water molecule into the mix, which sets up the transition to the actual signaling state of the protein.
If you want more details, the paper is open-access, and includes movie files of these transitions and much more detail on what's going on. What we're seeing is light energy being converted (and channeled) into structural strain energy. I find this sort of thing fascinating, and I hope that the technique can be extended in the way the authors describe:
The time-resolved methodol- ogy developed for this study of PYP is, in principle, applicable to any other crystallizable protein whose function can be directly or indirectly triggered with a pulse of light. Indeed, it may prove possible to extend this capability to the study of enzymes, and literally watch an enzyme as it functions in real time with near- atomic spatial resolution. By capturing the structure and temporal evolution of key reaction intermediates, picosecond time-resolved Laue crystallography can provide an unprecedented view into the relations between protein structure, dynamics, and function. Such detailed information is crucial to properly assess the validity of theoretical and computational approaches in biophysics. By com- bining incisive experiments and theory, we move closer to resolving reaction pathways that are at the heart of biological functions.
Speed the day. That's the sort of thing we chemists need to really understand what's going on at the molecular level, and to start making our own enzymes to do things that Nature never dreamed of.