Friday, October 16, 2009

Photophysics and GFP

Hi all,

A reminder that this week we're reading about photophysics. If everyone was able to post their reading on the blog as soon as possible that would be much appreciated - that way when we get to the tutorial we can discuss things that we found interesting about other people's papers, or things that we didn't understand.

This week I've chosen a paper about GFP (green fluorescent protein), for which the Nobel Prize in Chemistry was awarded last year for its discovery and development. The paper is titled 'Excited state reactions in fluorescent proteins' from Chem Soc. Rev., 2009, vol 38 p 2922- 2934 (If you're not at uni, you will be able to find the paper through the library or Web of Science).Whilst the paper at first glance appears to be a bit long, I think that the most interesting sections to us are the Introduction (which gives a good background) and section 3 - Photophysics and proton transfer in wtGFP. It turns out that the wild type version of GFP described in section 3 fluoresces much better than the chromophore of GFP on its own (described in section 2). Section 4 describes how things change when GFP is mutated, and section 5 is on 'second generation fluorescent proteins'. If you're running seriously short of time, I would at least recommend reading sections 1 (Introduction) and 3.

For those who aren't familiar with the terminology, 'quantum yield' essentially refers to the ratio of photons in to photons out. This is either expressed in percentages, or a decimal. A high quantum yield means that your molecule is very fluorescent and a high amount of light that goes in is converted into fluorescence. A low quantum yield means that most of the light that goes in decays non-radiatively, and most of the light energy is not re-emitted.

For those that are stuck as for where to look, some good examples of photophysics are molecules that use FRET, or the use of FRET to get information about your molecules of interest, biological molecules that fluoresce or absorb light, articles about the use of GFP (the one I have posted is mostly about energy transfer in the molecule itself) or in photosynthesis (I only discussed purple bacteria in my talk). Once you start looking, you should find lots of examples.

3 comments:

  1. I think what I'm about to explain will be old news to everyone but me, but I thought I'd share it just in case:
    Kristen's paper describes proton transfer (in wild type GFP) being redirected over "low short barrier H bonds". I wasn't sure exactly what Meech meant by the words in "" marks. I have subsequently worked out that when hydrogen bonds are shorter than normal (>or= 2.8A) & in the range of 2.55A, protons can move pretty freely between the two atoms and we accordingly call the bonds low barrier H bonds (LBHB).

    tada!

    Also: A whole issue of Chem Soc Rev devoted to green fluorescent protein!!! Sometimes I wish I was doing more of this..
    (that was serious, btw, in case anyone thought it wasn't.)
    Thanks Kristen.

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  2. Thanks, ack! I hadn't actually looked into that in depth, but you've worked it out for me :)

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  3. Oh boy...

    A particular interesting variant is the S65T/H148D mutant of GFP. In this protein, the proton transfer is so fast that it beats currently available resolution (<170fs). Meech's lab did some transient IR on this protein and found something interesting - there is no evidence of proton transfer AT ALL in the IR spectrum! This suggests that the excited state in this mutant has no barrier to proton transfer - that the electronic excited state is both a neutral and an anionic form, coupled by a common vibrational state manifold!

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