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".
Today the Nobel in Chemistry was awarded "for the discovery and development of the green fluorescent protein, GFP."
GFP, RFP and the derivatives thereof are massively important tools for biological research. The Academy notes that there have been more than 20,000 publications involving GFP since 1992. Personally I use GFP almost every day in my work.
GFP was isolated by Osamu Shimomura from the jellyfish Aequorea victoria. He was first interested in the protein aequorin which releases blue light in a calcium ion dependent way. However, the jellyfish glows green! In trying to explain this they isolated another protein that glows green under UV light. When they characterized these proteins they discovered that aequorin emits light that of a wavelength that GFP can absorb. This means that in the light-emitting organ of the jellyfish, aequorin emits blue light in a Ca2+ dependent manner that is absorbed by GFP which then emits the green light that we see.
The next subject to tackle is how does the protein become fluorescent. This was studied biochemically and structurally. The crystal structure was eventually solved which showed that the protein forms what is called a beta-barrel around the residues that make the actual chromophore (the bit that emits light).
The next step was taken by Martin Chalfie. Once the protein had been identified, and cloned (the DNA coding for it was put into an easily manipulated form) the question become, does GFP fold and become active on its own, or is there some specialized group of proteins in the jellyfish that are necessary to make it active?
Technical Note: Promoters
Genes aren't expressed randomly. Many are expressed in very specific patterns at very specific times. (pretty picture of a fruit fly embryo showing different levels of the expression of a few genes). We know this because we can put a marker gene behind the regulatory DNA (the promoter) coding for this expression and see when/where it is expressed. For many years this was done using LacZ/X-Gal in fixed cells. However, if GFP can be expressed in living organisms, we can put it behind promoters and see, by fluorescence, when and where that gene is being expressed.
Martin Chalfie worked on the worm Caenorhabditis elegans. It was known that certain genes were only expressed in certain cells because of their gene specific promoters. Chalfie expressed the GFP gene in E. coli and then in a few specific cells in C. elegans under control of a specific promoter. It glowed bright green only in the cells known to be expressing genes controlled by that promoter. This showed that there was no need for jellyfish specific enzymes to make GFP work. After that GFP was shown to functional in yeast, mammalian cells, flies and all sorts of other experimental systems.
Roger Tsien continued this work by looking at how GFP works and creating mutants with beneficial changes to the original protein. By expressing the protein in bacteria growing without oxygen they were able to show that the protein wasn't fluorescent, but would gain fluorescence upon addition of oxygen, showing that oxygen is the only requirement for the protein to be active.
Tsien's lab created mutations in GFP that helped make it a more useful research tool. They made it brighter, last longer before it breaks down (photobleaches), fold and become active faster, and have different spectral properties. The last once is especially important. Having a cell expressing GFP is useful, but having a cell expressing GFP and BFP (blue) or CYF (cyan) or YFP (yellow) is far more useful.
Tsien also worked on DsRed, a red fluorescent protein that was isolated from coral. This protein normally forms a tetramer (four copies stick together) which made it less suitable for tagging proteins since it would cause aggregation. Mutations were found that made it monomeric (mRFP) as well as brighter, more stable, folded faster and spectral variants including mRFP, mCherry, dTomato, mOrange, mBanana and others.
More interesting are specialized probes using fluorescent proteins for detecting changes in intracellular environment such as calcium or pH. There are variants that fluoresce one color, but change upon exposure to a certain color light to fluorescence a second color. These are very useful for looking at the displacement of populations of a protein. For example, let's say we're interested in a protein expressed on the cell surface. We express a fusion with a photoconvertable GFP. We take a picture showing it's distribution on the cell surface, then convert it to another color. We then wait a little while and take two pictures, one of the old protein and one of the new protein (old protein has been converted to a different fluorescent color, new protein has the original color). This lets us see how the new proteins are incorporated with respect to the old proteins.
There are forms of GFP that aren't active until they are exposed to a certain color of light. This allows us to focus on the movement of only the proteins that are localized to the spot we activate.
Calcium sensitive probes are used extensively in neurobiology. When neurons fire, calcium is let into the cells. If we have a fluorescent probe that reacts to calcium we can image firing events.
We can determine if two proteins are very close together using FRET (fluorescence resonance energy transfer). We label the two proteins differently, e.g. one with CFP, one with YFP. We excite the CFP with one wavelength of light and it emits of photon of another wavelength. That emitted photon is close to the excitation wavelength of YFP which can absorb that photon and emit a photon of another wavelength. That will only occur if the two proteins are very, very close together usually within 1-10 nanometer (1x10-9m).
We can bleach (use intense light to render the GFP non-fluorescent) a patch of protein and watch the movement of labeled protein back into the bleached area, or the movement of that bleached area around the cell.
One useful property of GFP is that it retains its fluorescence in fixed cells. If we have a labeled protein or proteins, we can just fix them and look at them without having to use antibodies or other fluorescently labeled molecules to label our cells.
One of the great things about yeast (the system I work in) is that GFP tagging proteins is really easy. I can add a tag to a protein in a week! People have gone and tried to tag every single yeast protein with GFP and look at the localization (sometimes it doesn't work, the GFP may interfere with the function of the protein killing cells or mislocalizing the protein). (UCSF)
People are trying to do the same thing in other systems such as the mouse brain (Genesat).
Thousands of researchers use GFP/RFP techniques every day to study everything from the variability of gene expression in yeast to how neurons develop new connections to how cells become cancerous. Live-cell imaging is very powerful technique that has profoundly advanced biology since GFP first became a viable tool a few decades ago. I should be thanking these researchers every day from making my work possible, and making biological imaging so beautiful.
a neuron
a moving cell
bacteria expressing different colors
Most information came from Nobel website.