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.