The Chapman Chronicles, vol IV: More on Fluorescence of Rhodopsin and T4 Lysosyme

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My avid readers of last week's blog will recall that I talked about the various states that rhodopsin cycles between during the signalling process, and I said I would elaborate this week with a few of Chris Schafer's interesting methods for measuring properties of rhodopsin using fluorescence spectroscopy.  For those less-than-avid readers who did not read last week's post (shame on you), I'll provide a brief background but encourage you to read last week's entry.  Chris is working on understanding the protein rhodopsin, which is found in the rod cells of the eyes and is responsible for low-light vision.  Rhodopsin binds a photosensitive ligand called retinal, which actually absorbs the photon of light and isomerizes, inducing a conformational change in rhodopsin and propogating a signal within the rod cell.  Two of the properties Chris was trying to measure were retinal uptake and release rates for rhodopsin; I'll address the cliffhanger I left in last week's comment.
To measure retinal release rates, Chris would expose to light, or photobleach, a sample of dark-state rhodopsin and monitor its fluorescence over time. Normally, tryptophans in an amino acid sequence will fluoresce at a certain wavelength, but in rhodopsin with a bound retinal, the tryptophans instead transfer this energy into the retinal, which fluoresces at a different wavelength.  Thus, by measuring fluorescence before photobleaching and at intervals after photobleaching, Chris could monitor the rate of retinal release by rhodopsin by measuring the amount of fluorescence in the tryptophan and retinal wavelengths and comparing it to his baseline value.
Chris could not measure retinal uptake directly because the apoprotein opsin has such a high affinity for 11-cis-retinal that it binds nearly instantaneously.  Instead, he measured it indirectly by placing his protein in an excess solution of 11-cis-retinal and photobleaching his sample; he could then derive retinal uptake rate by the rate at which 11-cis-retinal in solution competed out the All-trans-retinal bound to his protein.
In other news, on my last day I got to use the spectrofluorometer---not the really expensive one with a laser that they use to measure fluorescence lifetime, but the older one used to measure fuorescence.  The actual operation of the machine isn't too complicated---just fill a cuvette with your sample, place carefully in the machine, and wait as it measures fluorescence at a range of light wavelengths.  I didn't break it or mess up, which was exciting, but Amber needs to finish the rest of the samples before she can draw conclusions about the data collected.
In a final commentary about food, I patronized Little Big Burger on the waterfront yesterday.  The food was good, but order multiple burgers because they are small.
Overall, I was very satisfied with my senior project experience.  I think it accomplished all of its goals---I certainly learned a lot about the skills and critical thinking needed for science research, and I think I helped Amber make significant progress on this particular aspect of her PhD.

Photo guide:
3938: The a frozen bacterial pellet, containing a protein section that I tried to purify.
3944: The same pellet has been resuspended in a lysis buffer and placed in the French cell press, affectionately nicknamed the "french press", to lyse the cells and dissolve the protein.
3950: The cold room, where I tried to purify the dissolved protein from the dissolved everything else.
3953: The Dr. David Farrens, by Leonardo da Vinci.


I am glad you got to use a fancyish spectroflourometer

Thank you for the Advanced Chem shout out in last week's blog. We do have one spectrometer that can measure fluorescence, but I haven't found a good experiment to do with it...