Here’s something that many of us don’t tend to think about when we think about enzymes: vibrational energy. But it’s long been thought that anisotropic vibrational energy transfer (VET) plays a role in both enzyme active sites and in things like coupling to allosteric sites. Getting a handle on that, though, has not been easy – how would you pick out one particular part of a protein and suddenly increase just its vibrational energy?
Well, this new paper has found an ingenious way of doing just that. The group (a multinational effort from Germany, Canada, and Denmark) used an unnatural amino acid, the azulene analog of phenylalanine. Azulene is beloved by those who know it for its striking blue color and odd spectroscopic behavior, and that’s what’s being taken advantage of here. When the molecule is excited by 600nm light, it undergoes an unusual conversion from the singlet excited state, shedding all that energy into vibrational modes. In effect, when you do this to an AzAla-containing protein, you are quickly and selectively heating up that particular side chain, which you have placed wherever you choose in the protein’s structure.
Of course, to realize that last part isn’t necessarily trivial, and in this case the team used a genetic code expansion, evolving a new aminoacyl-tRNA synthetase enzyme to handle the unnatural amino acid. They were able to use this system to put the AzAla residue into a model protein for allostery (PDZ3) and see what happens when vibrational energy gets suddenly injected. As a sensor for that, they also incorporated azido-homoalanine (Aha) residues, whose side chains can be monitored by IR spectroscopy, into the peptide ligand for the protein.
When the team irradiated systems of this kind with 613nm light, monitoring the azide group by IR, they did indeed see an instant VET signal. The azide absorption shifted to a lower wavenumber in two different experiments, one where the AzAla was relatively buried and one when it was on the surface. The latter showed less of an effect, though, because some of the vibrational energy got transferred out into the water instead. It appears that the vibrational energy transfer happens on the order of picoseconds, which is what you’d expect.
So now that this technique is feasible, the next step will be to incorporate this donor/sensor pair into a wider variety of positions and proteins. Eventually, this will allow a map of vibrational energy transfer for a given protein – we’ll be able to see how this propagates, through what parts of the protein structure, and at what speeds. A key part of protein behavior, one that we’ve been fairly blind to, is now opening up – this and new spectroscopic techniques in the area are going to tell us a lot that we’ve never known.