Fluorescent Proteins

This is how I wanted the title for this post to look:

Fluorescent Proteins

                On Mini-Amedeo, I posted a story about fluorescent sushi.  While a little unsettling if you’ve never seen something like this before, I assure you that the technique used to make these fish fluoresce has been known for quite a long time, heavily studied and remains a popular scientific tool.  Yes, the fish are “genetically modified,” but that’s a fancy (and somewhat unnecessarily nerve-inducing) term that really means scientists have added one extra gene into the fish (See Central Dogma post for information on genes and proteins).  Genetic modification is not a new idea.  It’s been done since the dawn of agriculture, but that is a topic for another day.

                So what is this gene that scientists inserted into the fish?  The gene for Green Fluorescent Protein, also referred to as GFP.  Ooooh.  What an interesting and rather self-explanatory name!  As it implies, under the right conditions the protein fluoresces green. 

                In the 1960s, GFP was discovered in Aequorea victoria, which is a jelly fish found off the west coast of North America (Figure 60.1).  The gene to encode the protein was isolated from the jellyfish in 1992 – 1994.  Scientists were able to use known techniques to place the GFP gene into other organisms, who then express the GFP and – viola! –  we have glowing fish.  Or cells.  Or mice.  Yes, I’ve seen them all.

                Would you like to see what GFP looks like?  I hope you said yes because it’s coming in Figure 60.2 regardless!       It’s a very pretty protein.  The 11 diagonal bands represent 11 beta strands (Protein Folding post) that create a barrel shape with a hollow center.  An alpha helix, which is responsible for the fluorescent qualities, sits in the center.

                Knowing the structure of the protein allowed scientists to tweak it.  Instead of fluorescing green, a new version of the protein could fluoresce blue (blue fluorescent protein, BFP).  Another could fluoresce yellow (yellow fluorescent protein, YFP).  Very soon, an entire rainbow of colors was available to the scientific community.

                What were scientists doing with these proteins?

                In the Central Dogma post, I discussed the organization of a cell.  Let’s say you knew that a particular protein is present in your cell but you don’t know where in the cell the protein is found.  For example, does the protein stay in the nucleus?  Does it hang out around the plasma membrane?  Is it just all over the cytosol with no real localization?

                One way to find out is to create a long gene that encodes GFP and your protein of interest together.  Wherever your protein of interest goes, your GFP will go as well.  The two proteins are said to be “fused together.”  You can put this long gene into your cell, let it express the fusion protein and then see where GFP fluoresces.  Wherever the GFP is, so is your protein.  The schematic idea of this is outlined in Figure 60.3.  

Figure 60.4 shows you some real images of cells that I made a few months ago.  You can clearly see red fluorescent protein (RFP), GFP, and cyan fluorescent protein (CFP).  Each color represents a different fusion protein and is telling me where things are located.

This may seem trivial (or really cool!), but in reality the discovery of GFP and the subsequent tweaks that were made to it to create an entire arsenal of colors was huge to the scientific community.  There are other ways to determine what proteins are doing in the cell, but this way is so clean and simple compared to the others.  In fact, the contribution was so great that in 2008, Osamu Shimomura, Martin Chalfie, and Roger Tsien shared the Nobel Prize in Chemistry for their work on these proteins.

I met Roger Tsien in 2005 during my first year of graduate school.  At the time, I thought he made pretty colors and his research was interesting; today I realize just how much his work allows me to do my own.

NOTE: I chose to not discuss how fluorescence works in this post.  However, you will notice that the pictures of glowing sushi are all under a black light.  That is necessary.  All the cell images I show you in Figure 60.4 were also taken by shining a similar light on the cells.  Under what conditions things fluoresce is specific and sometimes requires specific light shone on them before they will glow.  If I do a post on fluorescence in the future, I will definitely link back here! 

NOTE 2: Incidentally, if you cook the fish, then it will no longer fluoresce.  The ability to fluoresce is dependent upon a properly folded GFP protein.  Cooking destroys the folding of most proteins.

REFERENCES

History of GFP, Nobel Prize: Roda. “Discovery and development of the green fluorescent protein, GFP: the 2008 Nobel Prize.” Ann Bioana Chem (2010) 396, pgs 1619 – 1622.

GFP Crystal Structure: Ormo et al. “Crystal structure of the Aequorea Victoria green fluorescent protein.” Science (1996) 273 (5280), pgs 1392 – 1395.

PDB ID code for GFP: 1GFL (www.pdb.org), images were rendered in PyMOL