Practical Fragments has a soft spot for new biophysical methods to identify fragments, many of which are given unfortunately non-descriptive initialisms. To a list that includes SPR, ITC, STD, MS, TINS, CEfrag, and WAC, we can now add Microscale Thermophoresis (MST), described in a new paper in Angew. Chem. Int. Ed. by Philippe Baaske and colleagues at NanoTemper Technologies as well as academic collaborators.
Thermophoresis, also referred to as the Soret effect, occurs when particles move in response to a temperature gradient. In this case, the “particles” are proteins, whose movements depend on size, charge, conformation, and solvation, and can be altered by factors such as ligand-binding.
In MST, a fixed concentration of protein is incubated with varying concentrations of ligand in small capillaries. An infrared laser rapidly heats a spot on the capillary, and an ultraviolet light source excites aromatic residues within the protein. The fluorescence in the heated spot changes as the protein moves along the temperature gradient. This movement is affected by ligand binding, and so measurements at different ligand concentrations can be used to construct a binding curve.
The researchers used MST to study the binding of ligands to several proteins, including ionotropic glutamate receptors (iGluRs), p38-alpha MAP kinase, thrombin, and even the calcium sensor Syt1. The dissociation values determined by MST were mostly comparable to literature values, and the researchers could also perform competition studies in which adding an excess of one ligand blocked a different ligand for the same site.
A nice feature of the technology is that, since it uses native protein, one doesn’t need to worry about the effects of immobilization or conjugation, factors that researchers using SPR, TINS, and WAC must consider. On the other hand, the fluorescence signal relies on native amino acid residues (tryptophan in the examples here), which can be obscured by many compounds. Also, in its current incarnation MST doesn’t appear particularly high-throughput, though it also doesn’t use much protein
Still, this seems like a pretty cool approach. I’ve started seeing NanoTemper at more conferences (such as FBLD 2012), so hopefully you will have a chance to check them out and let us know what you think.