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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: Twitter: Dereklowe

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March 7, 2013

Probing A Binding Tunnel With AFM

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Posted by Derek

UCP%20AFM.jpgEvery 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!

Comments (6) + TrackBacks (0) | Category: Analytical Chemistry | Biological News


1. Dave on March 7, 2013 8:48 AM writes...

The fact that they have done this with a membrane protein is particularly interesting to me - techniques like SPR screening are generally thought to be more challenging (though do-able) for membrane systems.

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2. Anonymous on March 7, 2013 8:56 AM writes...

These type of bio-analytical techniques can be quite sensitive and interesting. Once you think about what you can do with biotinylated pull-down assay, reversing the process and hooking it up to an AFM probe seems to be quite reasonable. I remember working on a design of a molecular probe that was suppose to clog the barrel of a translocating enzyme leaving something inside and outside of the central pore. The good old days ...

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3. anon on March 7, 2013 9:09 AM writes...

most of the trouble with membrane proteins is the isolation and handling without messing it up (and how do you tell that it is messed up). most of figuring that out is a black box of screening different lipids.

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4. Olivier Boss on March 7, 2013 9:43 AM writes...

Good job explaining these new data, Derek! These techniques are well beyond my expertise.
To put things in context, UCP1 is the mitochondrial membrane protein that enables brown adipocytes ("brown fat" cells) to dissipate as heat ("waste") a large part of the energy (from oxidation of fat and glucose). UCP1 uncouples oxidative phosphorylation, i.e., it is a regulated proton leak in brown adipocyte mitochondria. UCP1 is expressed only in brown adipocytes, no other tissue-cell type.
Olivier Boss (Energesis Pharmaceuticals)

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5. nitrosonium on March 7, 2013 11:00 AM writes...

always suspicious about AFM/chemistry papers.

what are the chances that the real system looks ANYTHING like the system depicted in the scheme?? even if it did, how does one unambiguously make that determination??

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6. student on March 10, 2013 5:40 PM writes...

AFM is pretty cool, it can make a video of myosin moving along a fiber
amongst other things (the DNA polymerase one is cool)

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