What happens when you chuck an active enzyme into the wrong solvent? Well, it stops working (or at the very least, it stops working as well as it did). And how do you know which one is the wrong solvent? Why, those are the ones that make it stop working.
That round trip is to illustrate that there’s often really no way to predict this sort of thing. The only way to know if a particular protein will still be functional in Buffer X or with a few per cent of Solvent Y is to check it and see. Better-known proteins have already had such work done on them, so the pH tolerance of widely used enzymes and their ability to work in (say) 2% DMSO is already established. But this is a pile of empirical data, every time, because we don’t have a very detailed picture of the ways that protein activity can go haywire as the solvent environment changes.
This paper (among others) is an attempt to bring some light into this area. It may sound odd or tedious if you’re outside the field, but this is yet another way to get more of a grip on the mighty Protein Folding Problem: given a particular sequence of amino acids, what three-dimensional protein structure can you expect to form? Living systems are exquisitely arranged around just that behavior; protein structure is a fundamental attribute of life. Our existence depends on these folding events being controllable and on repetitive, reproducible changes in protein structures. And in the other direction, there’s a lot of interest in getting enzymes and other proteins to perform under nonphysiological conditions and in nonaqueous solvents, for industrial uses and the like. So understanding what happens when a working protein starts to become deranged by solvent effects is a worthwhile approach.
In this work (from a multi-center team in Germany), the test enzyme is HMG CoA reductase, the famous target of the statin drugs and a very well-studied system. To give you an idea of how complex the situation is, adding any simple alcohol co-solvents decreased enzyme activity as the concentration went up, as you’d expect. Small amounts of DMSO, on the other hand, actually increased activity, although higher concentrations brought it back down. But DMF, also in the polar aprotic solvent category, hurt activity without ever helping it. Dioxane was the most destructive solvent for enzyme activity, but THF had hardly any effect (and in fact, up at 20% by volume, actually helped activity a bit). Acetonitrile was similar to THF; at 10% by volume it also increased activity. Acetone, though, was nearly as bad as dioxane.
Through small-angle X-ray studies, calorimetry, melting point shifts, and other techniques, the team was able to unravel some of the various mechanisms that lead to this suite of outcomes. The alcohol solvents, particularly ethanol, actually act as inhibitors in the active site as well as affecting the structure of the protein as a whole. On the other hand, the increased rate with a solvent like acetonitrile seems to be due to increased flexibility of the protein structure. You could certainly imagine that going either way, though – too much flexibility allows the protein to flop around to inactive conformations, whereas making a bit more flexible can be sort of “greasing” it for faster turnover. DMSO seems especially powerful for causing structural changes as you move to higher concentrations. These can occur as structurally important water molecules are stripped off the protein, or as solvent molecules themselves start making new (and destructive) interactions themselves.
On the other hand, dioxane’s nasty effects are apparently still a mystery, as are the differences between it and THF. In general, the paper concludes with one of those “Sure is a lot more work to do” situations. “The interactions investigated are often peculiar and, at the present level of knowledge, a holistic theory (based on first principles or empirics) cannot be inferred“, the paper says, and I can well believe it. This is a hard problem that gets into some fundamental principles, and it’s going to be a while before we really have a grip on it.