Studying membrane proteins is not easy. The broad scope of the problem clearly deserved a Noble prize in 2012. Thanks to these advances, today scientists can determine structures of some membrane proteins (e.g., G protein-coupled receptors). But some of them are so huge and complex that X-Ray crystallography and NMR spectroscopy don’t help.
Serotonine transporter (SERT) is one of such mules. The protein is an important target of antidepressants and (of course) recreational drugs. There was some controversy about how many binding sites the transporter has, one or two. And traditional means of experimental structure elucidation (crystallography and NMR) were unsuccessful. And computational modeling was of no trust for the team of Austrian biophysicists (Peter Hinterdorfer lab). So they called for additional Force, single molecule recognition force spectroscopy (SMRFS).
To study SERT, the authors covalently attached two enantiomers of citalopram to the tip of the atomic force microscope (AFM) cantilever. And they fished for the receptor on the surface of living cells expressing SERT. The binding of citalopram to the receptor generated some stretching (?) force when they were pulling the cantilever up from the cell surface. This allowed the authors to unambiguously identify two distinct binding pockets (orthosteric and allosteric).
As if it was not cool enough, the authors also performed a standard pharmacology study. They fitted AFM data to find koff of citalopram, nicely confirmed dependence of the orthosteric binding on Na+, and disrupted allosteric binding by point mutation in the pocket. Finally, they studied competition between their AFM-labeled citaloprams and unlabeled ones. From this experiment they could even see the positive allosteric cooperativity between enantiomers of the same configuration, but negative cooperativity for the opposite enantiomers!
I don’t know what to write in the conclusion. The science is just amazing!