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  • Hello, I'm Roger Tsien.

  • And I'm here today to tell you about fluorescent proteins.

  • which have made a major impact on microscopy

  • because they are genetically codable and

  • provide a relatively direct link from molecular and cell biology

  • into colors that we can directly see

  • particularly in the light microscope

  • but also, as you will see later on,

  • more macroscopic levels as well as eventually now

  • beginning to help us with super-resolution.

  • So, here this picture is, of course,

  • of the jellyfish, and

  • this jellyfish is where it all began.

  • And in a way, this is the creature that we most have to thank.

  • This is the jellyfish Aequorea victoria.

  • which was studied in Puget Sound

  • by Professor Shimomura, and in a moment,

  • we'll learn a little bit more about him soon.

  • This is the source of two of the most actually valuable proteins

  • in cell biology

  • First, there is the protein that actually enables this jellyfish to glow

  • And that's called aequorin, and

  • when the jellyfish is alarmed in the water,

  • and the water is disturbed, it emits a glow.

  • And then the partner of aequorin is the

  • green fluorescent protein, which changes the color from blue to green

  • to this day, we really don't have a well-accepted explanation

  • as to why the jellyfish wants to glow

  • why should it show this remarkable phenomenon

  • when the water's disturbed

  • nor do we know why when the jellyfish glows

  • why was it so important that it glows green instead of blue

  • why not just leave aequorin, or

  • if it really was important to glow green

  • why not just change aequorin in the first place to make it glow green directly

  • rather than invent an additional protein

  • but we are very grateful to the jellyfish

  • for having invented GFP.

  • So, the example - this is perhaps one of the only videos that we know of where we can show you the

  • glow of the jellyfish.

  • And this is a jellyfish upside-down

  • in a beaker in an aquarium at Alaska

  • where the jellyfish can still be found.

  • And we are illuminating with ultraviolet light right now.

  • And you may be able to see that there's a circle here.

  • There are little dots around the edge; that is the jellyfish glowing.

  • And this is the green fluorescent protein naturally made by the jellyfish

  • that we are exciting with the UV lamp.

  • I'm sorry the beaker is a bit fluorescent.

  • We should have got a better beaker that didn't have any,

  • but it's some yellow background.

  • But nevertheless, you can see the jellyfish there.

  • And it's upside-down and confined to this little beaker.

  • And what we're gonna do in a moment

  • is poke it to stimulate the flash.

  • So we're gonna start here, and you may be able to see

  • that the jellyfish is slightly moving around.

  • In this video, we are playing with the illumination

  • trying to get it nicely aimed at the jellyfish,

  • and in a moment, we're going to bring in this rod, with which we are going to poke it.

  • Then we turn out the lights, and we stir.

  • And during that stirring, that was the light emitted by the jellyfish

  • - not the light that previously had been applied from a UV lamp.

  • So that's it. That's the flash.

  • And maybe, you might have been able to see

  • it was sort of greenish - maybe.

  • Anway, the man who discovered that phenomenon is shown here.

  • This is Osamu Shimomura, and in 1962,

  • he published his first paper on aequorin.

  • And that was really the protein he was most interested in.

  • It had the really exciting job of turning chemical energy into light.

  • And he also mentioned that he found this contaminant

  • that got in the way a little bit of the purification

  • and that it was a greenish protein that fluoresced green

  • and that later he mentioned that it changed color of the jellyfish from the blue of aequorin to the green of GFP.

  • Later in 1979, he actually proposed the structure of the chromophore, GFP

  • based on surprisingly little evidence

  • but he got it almost absolutely, completely correct.

  • There were only very very small modifications from later work that came in.

  • This is a picture of him much later in 2008

  • in the rehearsal for the Nobel Ceremony.

  • And he is actually holding a UV lamp.

  • That's the violet that's on your left, and then next to it is the tube of GFP.

  • And this is the last tube in existence that is actually made from real jellyfish that he had kept in his freezer all this time.

  • Since then, everything has been made by molecular biology

  • and that's due to this man, here, Douglas Prasher at the center

  • of the photograph who in 1992 published the cloning of the gene for GFP.

  • And that was a long struggle. He didn't know what to look for.

  • And he could not predict that it would become green fluorescent.

  • In fact, that was a major worry

  • that there was no precedent for a protein that made by a biological organism would absorb blue light

  • and fluoresce green.

  • We didn't know whether it needed any cofactors.

  • Normally, in many cases, when biology does this sort of remarkable thing,

  • with light, it uses small molecule cofactors.

  • you're seeing me, for example, not because the proteins in your eye directly

  • absorb the light through the protein, but rather,

  • because the proteins bind retinal, the pigment that we have

  • to get from other sources, and that binds into the protein to make the functional protein.

  • And for all we knew, the fear was that maybe this protein used another cofactor

  • Or the chromophore structure that Shimamura had proposed might take many enzymes to generate out of the protein.

  • But, Marty Chalfie, who's shown here on the right side of the picture

  • then got the clone, the gene, from Prasher, as I did as well,

  • but Marty was first that proved that just that gene alone was

  • self-sufficient; in other words, if you took that gene

  • and expressed it in other organisms,

  • and he did it in E. coli and

  • in worms, then you would generate fluorescence.

  • And that proved that GFP was self-sufficient.

  • It knew how to make its own chromophore out of its own guts.

  • Fred Tsuji, not shown here, also did very very soon after Chalfie independently.

  • So, we now know that the chromophore structure

  • is something like this structure here

  • at the right, but it had to come from the

  • protein structure shown here

  • where just at this stage still amino acids.

  • There's a serine, a tyrosine, and a glycine

  • that somehow have to be modified,

  • and we have to introduce through a various number of steps,

  • which people are still now somewhat debating exactly how it happens.

  • We generated extra double bond; we generate a ring, and so on.

  • And all of this does require oxygen.

  • If you do not have oxygen in the atmosphere

  • when this protein is growing, it cannot become fluorescent or doesn't even absorb light - let alone become fluorescent.

  • So the oxygen is essential

  • and the only creatures which are biologically capable of using GFP

  • assuming we can get the DNA, the gene for GFP, in would be organisms that

  • cannot tolerate oxygen at any stage of their life cycle.

  • In other words, obligate anaerobes.

  • But they are a limitation. You do need oxygen.

  • So then, this may seem to be a completely unprecedented reaction,

  • but it turns out that the crucial first step is the attack of a backbone NH from Gly67

  • onto the amide bond of Ser65.

  • And this is a very strange reaction, you might think.

  • Since when does one amide attack another.

  • But in fact, it turns out to be fairly similar to a known

  • reaction in biochemistry

  • where the side chain amide of glycine attacks the side chain amide of Asparagine.

  • And that is promoted by bringing them closely

  • and it requires the special properties of glycine that it can curl up

  • and also that it's relatively unencumbered in its NH group.

  • And it's interesting that of all the fluorescent proteins that have ever been discovered,

  • the most conserved residue is not the one that contributes the most atoms to the chromophore

  • nor this other one that gets attacked,

  • but rather Gly67 - every known fluorescent protein has Gly67 in it.

  • And maybe it's because that's necessary for that first step.

  • Later on, the Asn-Gly goes into a different path.

  • It also forms a ring, but it spits out ammonia and so on.

  • And they diverge. Now there's one important other feature about this chemistry that I have to mention.

  • Oxygen comes in and at some stage pulls off the hydrogens

  • that were on the tyrosine

  • connecting the beta-hydrogens of the tyrosine that connect it to the rest of the chromophore.

  • So O2 pulls off H2, and the product is hydrogen peroxide.

  • H2 + O2 does not give you water in this case.

  • You have to balance the number of hydrogens and oxygens.

  • H2 + O2 gives you H2O2, which is hyrdogen peroxide.

  • So there is one molecule of a slightly toxic substance

  • that's generated for every molecule of GFP.

  • And that's sort of required by the conservation of mass.

  • It has been now directly verified, and it is a potential problem

  • that GFP is not totally totally safe for the organism,

  • and making a heck of a lot of GFP could generate one mole of hydrogen peroxide per every mole of GFP you make.

  • And you have to keep that in mind.

  • Now, fortunately, most organisms that have grown up in air has some defense

  • against a slow bit of hydrogen peroxide that's trickling out.

  • And we seem to be ok, but it's something that you have to keep in mind.

  • I should also say that in this original scheme

  • we propose that dehydration went before oxidation, and actually,

  • that's not quite so clear at exactly which stage the oxygen comes in and makes hydrogen peroxide

  • maybe a little bit earlier than I drew here.

  • So the original jellyfish protein actually was not very strongly fluorescent.

  • This dotted line here is the spectrum of the wild-type GFP.

  • And it has a big peak at around 400 nm - just below 400 nm.

  • In other words, the jellyfish actually GFP glows best in the UV.

  • And it only has a minor peak out here in the sort of green that gave it its name

  • and that's the one everyone wants to use.

  • Why the jellyfish deliberately crippled this protein

  • so that actually 5/6th of the time roughly, it is generating the UV,

  • and only 1/6th of the time, the visible, we really don't know.

  • Nevertheless, mutagenesis soon showed that particular mutations at Ser65

  • which you might think wasn't that close to the chromophore

  • though it contributed some of the atoms turns out to clean up the spectrum enormously

  • and get rid of this UV peak

  • and accentuate the visible peak

  • and also make the protein more stable,

  • and so these are the ones, the descendants of these improved proteins are the ones that

  • everyone uses nowadays including the very popular

  • so-called eGFP - e standing for enhanced,

  • which was this mutation S65T + a folding mutation to make it fold a little bit better.

  • Another feature I should say is that the jellyfish originally

  • grew and lives in Puget Sound or very cold water.

  • It never had any reason to worry about the ability to fold

  • at warm temperatures, but so many of us

  • want to work on mammalian tissue

  • at 37 degrees, this original protein just sorta collapsed

  • and basically wouldn't fold officially.

  • And a lot of mutagenesis of which this was just the beginning

  • then gradually made the protein able to tolerate warmer temperatures and fold more efficiently.

  • So later on, the crystal structure appeared almost simultaneously

  • from two groups in 1996

  • one of them was of this mutant S65T

  • and it showed a beautiful 1-stranded beta-barrel.

  • So the GFP is almost a perfect cylinder

  • made out of these strands that cross in this sort of helical lattice

  • around the outside, and then up the middle is

  • an alpha-helix that carries the chromophore.

  • And the chromophore in this model here is the bit in the middle that has the red and blue atoms that show up at atomic level

  • whereas everything else is just beta-strands or alpha-helix.

  • And this revealed why, in a way, no other enzymes were necessary.

  • They couldn't have been necessary because the chromophore's completely deeply buried inside this cylinder

  • so nothing else could have ever gotten that.

  • So it was essential in a way that the protein learned to do surgery on its own guts

  • and thereby generate the chromophore

  • in that chemistry I was just discussing where the hydrogen oxygen somehow has to get in,

  • hydrogen peroxide gets out.

  • Simultaneously, the other structure which was more of the original wild-type GFP

  • happened to be in a different crystal form

  • and showed that the GFP was also dimeric.

  • And that's another form of GFP, and actually,

  • there's an equillibrium between the two.

  • And it turns out that the GFP has a hydrophobic patch

  • where these amino acids mentioned here - three of them from each of the subunits get together

  • and this greasy patch enables dimerization.

  • And this is something to keep in mind again.

  • And one of the slight problems with wild-type GFP is that it likes a little bit to dimerize

  • And dimerization is generally bad when we want to tag a particular protein

  • because that forces the protein that we're really interested in, the partner, to also become dimeric

  • when it wouldn't otherwise have been.

  • We have forced it to be dimeric by fusing it to GFP.

  • And that often changes the cell biology and messes things up.

  • It doesn't matter so much if you're just trying to light up a cell

  • and you're using isolated protein because

  • then the fact that it wants to be a dimer, if it's not stuck to anything,

  • nobody else cares, so to speak.

  • Nevertheless, there's been a lot of interest in the dimerization, and we now know exactly how to get rid of it.

  • For