Transcription and Translation

From the Nobel site:

8 October 2008

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2008 jointly to

Osamu Shimomura, Marine Biological Laboratory (MBL), Woods Hole, MA, USA and Boston University Medical School, MA, USA,
Martin Chalfie, Columbia University, New York, NY, USA and
Roger Y. Tsien, University of California, San Diego, La Jolla, CA, USA
“for the discovery and development of the green fluorescent protein, GFP”.

Well I certainly nailed this one. In fact I got up this morning thinking, “let’s find out if Tsien got the Nobel”.

This is a well deserved prize. Flip open any biomedical journal and you’ll see why – Green Fluorescent Protein (aka GFP) is probably the most used gene in the world.

It is safe to say that you can clearly divide microscopy into two phases, before GFP and after.

GFP is truly a wonder protein. If you excite the molecule with blue light it will convert this to green light which it emits. By monitoring the fluorescence you can pinpoint where GFP is located. Attach GFP to your favorite protein, for example a gene involved in cell duplication, and now the location of your fusion protein can be monitored inside off a cell. You might for example find out that your protein is localized to the chromosome during cell division and thus probably has a role in how duplicated chromosomes are pulled apart during mitosis.

Before GFP, we could only deduce how molecules and proteins were organized within cells after biological samples were fixed, extracted and stained with some sort of probe, usually a fluorescently conjugated antibody that recognizes the protein of interest. Wherever you detected fluorescence, you could assume that there was there was antibody bound to your fixed protein. Since the cells are fixed, these pictures were static. You knew where your protein might be, but had no clue how your protein moved around the cell.

After GFP, we can now deduce how molecules and proteins were organized within biological samples in LIVE CELLS. This has three advantages.

1) The samples do not have to be fixed and you don’t need antibodies. Sometimes fixation disrupts cellular organization, sometimes antibodies cross react to other proteins. These problems do not exist with GFP technology.

2) We can now deduce the BEHAVIOR of proteins.Think of it this way. Before you only had still photographs. Now you have movies. GFP technology literally provides us with an extra dimension of information (in this case time). Understanding how proteins move around inside a cell gives us a tremendous amount of information about how cell organization is achieved.

3) GFP has allowed us to develop all sorts of tricks.

Through the use of a technology called two-photon microscopy, biological samples can now be observed from a distance. We can now observe GFP-labeled cells deep within a tumor or deep within the brain.

Destroy all the GFP fuorescence in one area of the cell, and now you can detect how GFP-tagged molecules from outside this area diffuse back into the area – you now just measured the diffusion rate of your protein of interest, this is called FRAP (Fluorescence Recovery After Photobleaching).

Here’s another trick, have protein X tagged with yellow fluorescent protein (YFP) and protein Y tagged with cyan fluorescent protein (CFP) – if proteins X and Y interact inside of the cell, the CFP will activate the YFP. You now have a technique called FRET (Fluorescence Resonance Energy Transfer) that allows you to monitor if, how and when two different proteins interact within live cells.

And there’s more. GFP has been modified by researchers such as Jennifer Lippincott-Schwartz, so that GFP can be activated and turned off. This has allowed researchers to monitor the half-life of proteins. It has allowed researchers to see how proteins redistribute from one area to another. Other GFPs have been modified so that they fluoresce differently depending on pH. In one of the most amazing papers ever, Roger Tsien used another fluorescent protein, called dsRed and literally evolved the protein into other fluorescent proteins that cover the entire spectrum of colors.

i-436cc1f240fcc92ca7d627d73be8dcda-tsien2.gif

I’ll post something latter this week on this remarkable paper.

Here’s a link to a lecture given by Tsien at the Nobel Symposia

Comments

  1. #1 ponderingfool
    October 8, 2008

    Here’s another trick, have protein X tagged with yellow fluorescent protein (YFP) and protein Y tagged with cyan fluorescent protein (CFP) – if proteins X and Y interact inside of the cell, the CFP will activate the YFP. You now have a technique called FRET (Fluorescence Resonance Energy Transfer) that allows you to monitor if, how and when two different proteins interact within live cells.
    *************
    You can also test protein-protein interactions using split GFP. Attach one half of GFP to protein X and the other half to protein Y, if they interact the split GFP halves come together and you have an active GFP that fluoresces. A very cool enzyme to say the least.

  2. #2 joe
    October 8, 2008

    Yeah, yeah GFP is great (no doubt) — but it has also lead to the greatest amount of self-delusion in biology. Don’t forget that GFP and YFP are both highly pH sensitive — and pH varies wildly locally at the level of molecules (inside cells, we’re not talking about homogeneous bulk solutions).

    On top of that, FRET is often done terribly, since few understand the sensitivity of the technique to sub-Angstrom variations in position and orientation, or how to do it properly for high temporal/spatial resolution.

    I’m sure we’re getting lots of data that will be found in ten or twenty years to be all wrong.

  3. #3 Alex Palazzo
    October 8, 2008

    Ponderingfool,
    Yeah the split GFP is a great technique, however it doesn’t always work, you need some luck in that the two GFP halves need to be in the right conformation inorder to produce a bioactive molecule. I’ve written about this before here:
    http://scienceblogs.com/transcript/2006/04/bimolecular_fluorescence_compl.php

    Joe,
    I 100% agree FRET has been used poorly by most researchers. The best FRET I’ve seen is whole spec FRET where the complete spectrum is analysed in a cuvet so that you can precisely measure the energy transfer between the two fluorophores – of course this can’t yet be performed with a microscope. I’ll just point you to this long rant of mine on FRET from last year:
    http://scienceblogs.com/transcript/2007/05/all_you_microscopist_wannabes.php

  4. #4 Arikia Millikan
    October 8, 2008

    Yay! I *heart* GFP! I used to make mouse 293-T stem cells glow with it in the Radiation Oncology lab.

  5. #5 Martijn
    October 8, 2008

    You can also do FRET and FRAP at the same time and see whether the mobility of the interacting molecules is different from the non-interacting ones, like my colleague Martin van Royen did. I would advice against it though if you have less than, say, two years time.

  6. #6 SJ Green
    October 8, 2008

    Indeed, well deserved. His contributions are reflected (no pun intended) in both his publications and through his extensive co-author/collaborative network as captured in his interactive profile at pubScholar:
    http://www.pubscholar.com/authors/profile/roger-y-tsien/publications

  7. #7 Jim
    October 8, 2008

    I agree that most papers that rely on FRET to determine protein-protein interactions leave a lot to be desired, but there has recently been a lot of work using photoactivable fluorescent proteins and internal reflection microscopy that give much better results. Do a PubMed search for fPALM for some really interesting stuff that can be done with fluorescent proteins.

  8. #8 Geroge Smiley
    October 8, 2008

    I’m ambivalent about Tsien sharing the prize. True, Tsien has done the most work, but he did not make the key discoveries. Very many people besides Tsien have also done a tremendous amount of work to make improved fluorescent protein variants, notably groups at RIKEN but also many others.

    Shimomura is the one who discovered the thing. Prasher cloned it. Chalfie used it as a tool.

    It is true that if you add in Tsien’s work on small molecule sensors, particularly small molecule Ca2+ sensors, that his contibution is monumental. But Nobel Prizes are generally given for specific advances and not for lifetime contributions. Just my $0.02.

  9. #9 Tor
    October 9, 2008

    cool! I just learned about GFPs in my cell bio class today…freaky…and humbling how much I still have to learn.

  10. #10 SJ Green
    October 9, 2008

    Geroge, your points are right on, however, it took the individual contributions of all 3 (and, as you reminded us, the efforts of Prasher) to advance the impact GFP has had; I think the Nobel statement captures it correctly: “… rewards the initial discovery of GFP and a series of important developments which have led to its use as a tagging tool in bioscience…”

    Collectively, their efforts were critical to advancing this and it is equally interesting to see the vast number of collaborators/coauthors that they worked with over the years as refected in pubscholar:

    For Chalfie -
    http://www.pubscholar.com/authors/profile/martin-chalfie/collaborators

    For Shimoura –
    http://www.pubscholar.com/authors/profile/osamu-shimomura/topics

    For Prasher -
    http://www.pubscholar.com/authors/profile/d-c-prasher/compounds

    And Tisen as discussed previously.

    Well deserved and properly shared by all.

  11. #11 wesele
    November 2, 2008

    Anyway, I’ll certainly be quoting Powel in my letter to Hagan. I urge every atheist (or even theist who believes in civil rights) to do the same.

    ~Javier

The site is currently under maintenance and will be back shortly. New comments have been disabled during this time, please check back soon.