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March 7, 2013
Probing A Binding Tunnel With AFM
Every so often I've mentioned some of the work being done with atomic force microscopy (AFM), and how it might apply to medicinal chemistry. It's been used to confirm a natural product structural assignment, and then there are images like these. Now comes a report of probing a binding site with the technique. The experimental setup is shown at left. The group (a mixed team from Linz, Vienna, and Berlin) reconstituted functional uncoupling protein 1 (UCP1) in a lipid bilayer on a mica surface. Then they ran two different kinds of ATM tips across them - one with an ATP molecule attached, and another with an anti-UCP1 antibody, and with different tether links on them as well.
What they found was that ATP seems to be able to bind to either side of the protein (some of the UCPs in the bilayer were upside down). There also appears to be only one nucleotide binding site per UCP (in accordance with the sequence). That site is about 1.27 nM down into the central pore, which could well be a particular residue (R182) that is thought to protrude into the pore space. Interestingly, although ATP can bind while coming in from either direction, it has to go in deeper from one side than the other (which shows up in the measurements with different tether lengths). And the leads to the hypothesis that the deeper-binding mode sets off conformational changes in the protein that the shallow-binding mode doesn't - which could explain how the protein is able to function while its cytosolic side is being exposed to high concentrations of ATP.
For some reason, these sorts of direct physical measurements weird me out more than spectroscopic studies. Shining light or X-rays into something (or putting it into a magnetic field) just seems more removed. But a single molecule on an AFM tip seems, when a person's hand is on the dial, to somehow be the equivalent of a long, thin stick that we're using to poke the atomic-level structure. What can I say; a vivid imagination is no particular handicap in this business!
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