My photophysics reading for this week has been "Quantitative FRET Analysis With the E
In this article, some scientists from various institutions within the Scuola Normale Superiore in Pisa propose a new fluorescent protein pair (E0GFP-mCherry) which they argue is particularly useful for FRET experiments in living cells.
The link here to Kristen's paper should be obvious and, in case you're interested, I got a bit carried away trying to find a link to Michael's. I might have come up with a link that actually has some conceptual substance by Tuesday, but in the meantime here's my best effort: mCherry is a monomeric derivative of DsRed, a red fluorescing protein derived from stony coral. Michael's paper features HcRed, which is also red fluorescing and also derived from an anthozoa, but is dimeric & found in nature in an anemone, not a coral.
back on track... my article describes the quantitative determination of FRET efficiency for this pair using fluorescence lifetime imaging spectroscopy (FLIM) & acceptor photobleaching (APB). I found the format a bit "biology-esque" (with descriptions of method focussing on technique used for, say, cloning proteins) but these were all new ideas to me so it was interesting and I coped! I recommend skipping to the treatment of FLIM & APB in the Results section which was somehow more conceptual.
The authors also talk about "the recent phasor approach" to FLIM, which I had to go here to understand.
I'm sure you don't have to come up with a link to *everyone* else's paper - if you could do that, then it means we've all looked at really similar aspects. Diversity is better :D
ReplyDeleteAlso, your links don't work properly - the website of the blog is in front of the actual address you want. So you want to go to http://dx.doi.org/10.1111/j.1751-1097.2008.00435.x for the paper and to here http://dx.doi.org/doi:10.1529/biophysj.107.120154 for the phasor approach explanation
ReplyDeleteOooh... I hadn't actually heard of E0GFP before this, Ack. Many thanks for bringing a new variant to my attention! Looks like a nice example of a dominant A-band (neutral chromophore, according to the dominant wisdom) green FP. Interesting mutations - I'll have to refresh my memory of where position 231 falls in the wild-type structure. My guess is that it may be near the chromophore, because having L instead of H might mean getting rid of a compensating charge that would stabilize the anionic chromophore form. Position 64 is right upstream of the chromophore, and I imagine that getting rid of a polarizable F residue also destabilizes the anionic form...
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