(Note: a lot of what’s written below are from rough notes quickly taken during the sessions. Any inaccuracy is regretted, and I will be happy to correct any mistakes I made in either interpretation or presentation of the talks)
The day started out quietly, collecting the badge and other registration materials – including a very cool water bottle to make sure you remain hydrated through the grueling five days – followed by a great breakfast at Miss Shirley’s Place (highly recommended, especially the crab Benedict). But due to the quirks of the subgroup meeting scheduling, the afternoon turned out to be a blur of activity – flitting between four rooms, making sure I took it in all the science.
Most of the talks I attended today, spread between the Biological Fluorescence, Membrane Biophysics, Membrane Structure & Assembly and Molecular Biophysics involved fluorescence in some manner. Claus Seidel talked about how correlating various Forster’s Resonance Energy Transfer (FRET) measurement parameters (lifetime, fluorescence quenching and polarization) lead to richer information. His group has also developed software for using FRET distance constraints for developing structural models. This seems quite cool, since FRET has mostly been associated with comparison of structures rather than absolute measurements. But I would be interested in how all this is applied in protein systems where the change of conformations are often quite subtle (the talk was based on RNA and DNA four way junctions).
Dynamics and fluorescence measurements were the theme of majority of the speakers at the afternoon Membrane Biophysics subgroup symposium. Prof Scott Blanchard spoke of their use of single molecule FRET in investigating substrate modulated gating dynamics in a model transporter, LeuT. They identified sites on both the extracellular and intracellular sides of the protein that under large conformational changes detectable by FRET at the single membrane protein levels. Through experiments on the wild type and a gating mutant and by using substrates and non-competitive inhibitors, they provided structural evidence for the alternative access model of transporters. According to this model, transporters are conformationally open to one side of the membrane at a time only, and substrate binding lowers the activation energy barrier for the transition between the outward facing and inward facing conformations. This alternate access model seems to be a continuation of a theme from last year – as more and more experimental and simulation data are pouring in its favor. Personally, this was rather exciting as I had worked on a similar (but a bit more complicated) transporter protein during my graduate school and part of my thesis involved proving this in vs out conformational change (albeit at a much lower resolution than achieved by such single molecule methods).
While these experiments were conducted with proteins in detergent micelles outside their natural environment, earlier in the session, Baljit Khakh from UCLA presented data on single molecule tracking directly in cultured cells – by using cell-injected engineered P2X receptor proteins that could be labeled with Quantum Dots (QDs). P2X receptors are important in variety of physiological roles, including – as the speaker pointed out – the rather non-trivial function of bladder release. Plus, it is involved in neuropathies in microglial cells. The labeling of these proteins with QDs offered various advantages for in situ tracking, including longer lifetime, brightness and the blinking (this property can be used to confirm single molecules are being tracked). Combining this imaging property with whole cell patch clamp, their group have made important stride in understand the correlation between the protein mobility and ATP induced currents produced by the channel. For example, they demonstrated that Ca2+ ions are important for the mobility of the P2X4 receptors, and that such mobility was highly subunit and cell dependent. Of course, this isn’t the full story, and the group is pursuing further studies, which includes amongst others, trying to image single channel mobility while measuring its current activity simultaneously.
Simultaneous measurement of current and fluorescence dynamics was the theme of two other talks in session. Peter Lu from University of Bowling Green, OH, demonstrated the ability to measure simultaneous fluorescence (FRET) and electrical signals. They used two model systems – gramicidin channel s and Colicin Ia – on their platform to correlate structural changes to the function of these ion pores.
Mark Wallace of the University of Oxford is also building a system for the same purpose, but employing a novel platform that utilizes an extremely stable planar lipid bilayer formed between a oil-droplet and an agarose hydrogel. The non-specific bacterial protein pore, alpha hemolysion , was used as an example to demonstrate correlated electrical signals and fluorescence measurements on the droplet bilayer.
Hopefully, all these platforms can be used for exquisitely sensitive studies correlating the structure and function of a wide variety of membrane proteins in future. As many biophysicists are aware, membrane proteins represent a significant portion of the human genome and are involved in number of pathologies, but are still very poorly studied. Part of this is due to the difficultly of purifying the proteins and obtaining high resolution crystal structures. The latter problem seems to have been solved to some extent in last 10 years or so which have seen an explosion in high-resolution structures of membrane proteins. However, such crystal structures are only part of the full story – these are highly dynamic entities (as seen in many of the talks today). Application of smFRET methods, which have yielded a wealth of information on nucleic acid dynamics in the last ten years, combined with electrophysiology data and theoretical approaches such as MD simulations should go a long way in figuring out how these molecular systems work.
The meeting kicks into a higher gear from tomorrow (Sunday, March 5th) with the full plethora of platform sessions, symposiums and posters. Not to mention the exhibitors (ie lining up to grab that free t-shirt from Avanti!). I will check out the dedicated blogger’s computer at the BPS office tomorrow – that might help in more regular updates rather than a single data dump every night (though I am still a bit miffed by the lack of wireless access at the convention center).