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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 example, if you take the A206, which is one of the ones mentioned
and change it to a lysine, A206K
completely destroys the dimerization because
then a lysine and a lysine replacing the alanine and alanine are now
in each other's face, and the electrostatic repulsion
between the two plus charges blows apart this dimer interface
and we supress the dimerization.
So in anytime that you are in any concern about it, there are mutations that can be used
to fix the dimerization problem, and they don't seem to have any bad effects on any of the other properties of GFP.
So can we get other colors?
And there was a lot of interest in getting other colors for various reasons.
The very simplest is that sometimes we want to follow several different proteins simultaneously
or follow different cells, which are marked with different and by making them different colors,
we can distinguish all of these.
So the original color is pretty close to this - the green output
if you forget the UV that the wild-type produces, you wouldn't have seen it with your eyes anyway
the residual green is very similar to this green from S65T
except S65T puts all of its energy into this green instead of only 1/6th of it.
It turns out to be possible to make a blue, a cyan, and evena somewhat yellowish version.
It's not awfully yellow; it's a yellowish-green still. But we call it YFP.
And these were found by various substitutions of the chromophore or around the chromophore.
So for example, if you change the original tyrosine in the chromophore, which is here,
and change it to a histidine, then we get the blue.
If we change it to a tryptophan, we get the cyan.
Ánd by the way, the UV peak was when this tyrosine was neutral.
And the green peak was when the tyrosine is negatively charged.
And this turns out to be crucial for my next lecture where we often, people now play with this equilibrium
and make it subject to environmental influences.
And by changing the ratio basically of the UV to the visible peak, we can make a GFP that switches on due to environmental influences.
And finally, the yellowish version is by taking an amino acid that's remote in the primary sequence that's
threonine 203 changing it to things like tyrosine or phenylalanine
and thereby it's close in 3-dimensional space.
It stacks above the real chromophore, and
the pi-pi stacking is what we believe is responsible for the yellow shift
that makes it a somewhat yellowish-green.
If we now look at the range of fluorescent proteins up to now,
everything was based on GFP from the jellyfish with mutational engineering
but it turns out that fluorescent proteins are fairly widely distributed in nature.
In fact, there are homologs in vertebrates
though they happen not to be fluorescent
if they were, we'd all be walking around like sort of the green giant.
But in fact, the ones that are all fluorescent all come from
the phyllum Cnidaria
which is the glorified Latin name for basically the family of coelenterates that includes
jellyfish, and corals, and some other related organisms.
And it's not just the jellyfish that can make fluorescence, but it turns out that corals
and that was the brilliant discovery of a Russian group, first author Matz et al.
And they have subsequently have done a lot of the phylogenetic tree by doing this classic molecular evolutional and genome analyses.
So a lot of you know, of course, tropical corals have beautiful colors.
Every scuba diver knows that or even snorkeler
but what we didn't realize is that so many of those colors are actually due to
homologs of GFP,
and particularly, one that attracted a lot of interest and was the subject of that first paper by Matz et al.
was Discosoma, which has a beautiful red fluorescence.
And that paper was about the cloning of the gene
for a red fluorescent protein that they called Ds for Discosoma - DsRed.
And it proved to be this beautiful red color.
At that time, it was not clear right away how did the corals manage to coerce the chromophore into absorbing green light
now instead of blue
and fluorescing red instead of green.
But chemical analysis of the chromophore eventually showed
that was the corals had figured out was add an extra double bond, which is between the carbon in this place and the nitrogen here
and this forms a very unusual structure called an acylamine
that is basically completely unstable
in ordinary solution and is only held together
by virtue of being inside the protein
but that extra double bond here in addition to all the double bonds that were up here
now extends the chromophore and it recruits this ketone or carbonyl group that used to be part of amide
and this extended chromophore then turns out to beautifully explain the red color as even shown by quantum mechanical calculations.
And when this structure was determined, some people in my lab were really
pleased with themselves, and they used the actual bacteria expressing the protein and used this ink, and with little toothpicks, they drew the structure on a petri dish.
So you can both see the beautiful red color and the structure that gives rise to to it all in one picture.
Now DsRed made by the corals as typical was made by the coral for its own reasons
and to this day, as usual, we really don't have a definitive explanation that everyone accepts
for why the coral should want to have these colors.
But whatever it is, the protein was not ideal for cell biological use.
The biggest problem was that it turned out to be a very tight obligate tetramer.
GFP was a weak dimer, and even the weak dimerization gave problems.
But DsRed being a tight tetramer often prevents proper trafficking, or fusions.
It's even worse because it's really strong and tight,
and four copies of your protein that you're trying to label red
now get fused together
And an example here was connexin-43, which is a constituent of gap junctions
and its detailed structure is shown here.
If you fuse it to GFP, you can get a fluorescent gap junction
and it trafficks reasonably well and makes this fluorescent plaque in a micrograph, of course,
showing that there's a boundary between one cell and another
which is slightly fluorescent, but when you do the same with DsRed,
the plaque is of course trying to make this connexin-43 wants to make a hexamer on its own.
Two of them eventually get together to 12-mer or decamer; meanwhile, the connexin-43 is trying to make a tetramer.
The two clash with each other, and you get a mess.
And the protein can never make a gap junction.
There are additional problems like the DsRed took a really long time to turn red,
and it didn't finish the job.
It left some green behind.
But the fact that it went through green stage is an interesting clue.
Eventually, these were all fixed by mutagenesis.
It was much more difficult than with GFP because that tetramer was really hard to break,
But eventually now, we now have a whole gamut of different colors.
of fluorescent proteins derived from DsRed through monomeric forms,
and it turned out to be not too hard to change their color.
So over here are the four that came from the jellyfish -
blue, cyan, green, and yellow
and these are all, by the way, these are all little tubes of protein made in E. coli and purified and simply a photographed by their own fluorescent light
and then we eventually got these additional colors in my lab
and in order to separate them and keep them easy to remember, we gave them the names of fruits
which each color references. Like this one is honeydew.
because honeydews are sort of yellowish green.
And then there's tangerine and strawberry and cherry and so on
- all these different beautiful red colors.
The one exception that really isn't monomeric is so-called tandem dimer Tomato.
And that is actually two copies of the original 11-stranded beta-barrel that are permanently genetically fused to each other.
And in that way, they satisfy each other.
This protein is not a tetramer; it's a dimer. But it's internally satisfied.
It's got two copies, and when you fuse it, you fuse both together as one unit.
And that's why we call it a tandem dimer.
So I wanted to mention then, derived from these new coral proteins, there have been
actually there's some varieties that were made from original jellyfish as well.
It now turns out that there are proteins that can be deliberately changed with light.
Of course, some proteins bleach, and that can be very useful in techniques
like fluorescence recovery after photobleaching.
But an even more spectacular case is this protein called dendra
which starts out green like DsRed.
But whereas DsRed spontaneously changes from green to red,
this one sits green indefinitely until you shine blue light on it
and that is what switches it from green to red.
So we can use this as an optical highlighter
to, for example, in this case track the migration of
a protein called fibrillarin
these authors fused Dendra to fibrillarin and locally
turned, shined blue light on, turned some of it from green to red and then watched
the subsequent fate of just those proteins that had been labeled.
So those proteins that were in the spot first lost their green color - not quite completely.
It might have been better if it had been complete, but they lost it considerably.
And then there's some recovery as proteins move around into this illuminated zone.
And meanwhile, the red protein made in the spot then over time spreads out.
And we can watch. And these are 1, 2, 3, 4 are different regions of interest in the original that were being tracked.
And even more spectacular form of photochemistry is shown by Dronpa
which is reversible, and it starts out green fluorescent,
and as you continue to excite its green fluorescence using blue-green light,
it fades away and reaches essentially down to nothing.
And you might think that this protein is dead, and most fluorescent proteins had you done it, you'd be right.
But this one, when you shine violet light on it, springs back to life
and comes back fully completely, just about, rejuvenated.
And we can do this cycle after cycle after cycle, and we show here,
these are compressed time course of probably 50 cycles,
and there's a slight bit of rundown as you go through many many many cycles.
But you can do this over and over again.
And the applications for repetitively following movements inside cells or as you will later hear from other people for doing super-resolution microscopy.
There are major implications that have been exploited, but I don't have the time to go into them here.
But the authors were so pleased with themselves that they
simply laid down a film of protein, a wet film of protein
and then they shined its own name successively using this trick of
starting fluorescent, bleaching it completely, shining the letter D,
a silhouette of the letter D,
onto this film of protein, and then they
lit up the letter D, and then they erased it again.
and then they wrote it again, this time the letter R, et cetera and wrote out its name.
Ok, so I don't have time talk more about these very interesting photochemically active proteins.
I'm now gonna turn briefly to the question of how do we look inside a more intact animal.
especially animals that are not quite as transparent as the ideals.
Now, the ideals may be zebrafish and a lot of microscopists like zebrafish or C. elegans because
they are transparent, but many of the other important ones like mice or flies are somewhat more opaque.
And in general, most organisms, especially mammals are nearly completely opaque
down in short wavelengths
below 600 nm, and that's because of the peaks of hemaglobin.
After all, that's what makes us pink.
why flesh is red, and as long as it's there,
you can't easily excite through much thickness of this tissue.
A standard demonstration is that if I try to shine a green laser through even the thinnest of my fingers,
nothing will get through, but if I shine a red laser,
laser pointer, a lot will get through,
and that's because a red laser is about 630 nm
beyond the shoulder, so it would be really nice
if we had fluorescent proteins that could be both excited and would emit longer than 600 nm.
And the record for current proteins derived from the jellyfish, coral, cnidarian family is just barely over 600 nm
and sort of marginal.
But we'd like to get out say to the 700 or so.
And it turns out that this is possible, but you have to be willing to go to a whole new protein family.
And this protein family is now back to cofactors.
These are small organic molecules.
This is what we were afraid the jellyfish and corals were using.
Now, we actually use them, and these pigments are derived
from the heme biosynthesis pathway.
Particularly, this is heme with a porphyrin, which is four pyrroles in a big circle
and with an iron in the middle
and when we break down heme or any organism that uses heme, that's essentially the entire kingdom of life - almost
when we break it down with an enzyme called heme oxygenase, the first breakdown product is called biliverdin.
Biliverdin is something that every adult human being makes about a quarter gram of every day
Normally, it's sort of kept sequestered, but if any of you have had a big bruise,
and you turned a sort of black and blue, a lot of that color is broken down heme that makes biliverdin.
And later bilirubin here, which is part of what gets excreted.
So it turns out that weak biliverdin is phylogenetically ubiquitous
Some organisms elaborate it into known fluorescent pigments that have these more elaborate cofactors.
But it turns out that bacteria use bilverdin in proteins that sense light.
And by mutagenesis, it was possible to prevent them from sensing light and instead use the energy to fluoresce.
These now make infrared fluorescent proteins that absorb at 680-some nanometers and emit just over 700 nm, hence, their name as infrared fluorescent proteins.
And they are genetically encodable; you have to make sure that the organism either provides biliverdin,
or the cell provides biliverdin. And as I said, most of our cells make it.
If necessary, you could can supply the extra by injection if you don't think they make enough of their own.
And so up to now, this has been the only way to decisively get beyond 600 nm.
And an example of its use here is when this is transfected into the liver;
now we can see the liver through the skin of an intact mouse
Far far better than even a relatively red-shifted protein that was derived from corals
And in turn, that is better than GFP,
which is essentially hopeless.
You cannot see the liver
of a corresponding GFP-transfected mouse that's got that.
Instead, all you see is the autofluorescence or the fur around the outside.
Whereas this stuff is shining through the abdomenal cavity.
And there's many things more that need to be done to improve this relatively recent work.
There's actually thousands of phytochromes; this is extremely widely diversified gene family.
There may be possible to get longer than what I just described.
And it should be helpful for lots of interesting in vivo macroscopic fluorescence imaging
somewhat similar to microscopy but at a more a less-space resolution but greater depth inside intact animals.
It provides colors that are orthogonal even to the wide range we've already got.
It could be an acceptor for fluorescence resonance energy transfer if it's quantum yield was improved a bit more.
It may be also good for the photoacoustic type of imaging
and many other tricks, and finally if heme oxygenase activity is itself biologically important in a lot of metabolic activities,
it's responsible for making carbon monoxide and helps makes cyclic-GMP,
and this is a possible way we could detect that activity in a cell that doesn't have much of its own and doesn't have any other source of biliverdin.
So in conclusion, these are some of the people in my lab who contributed or that we collaborated with
who contributed most to it,
and once again, I'm showing you the fun that you could have with these multi-colored living inks made out of fluorescent proteins in which some people in my lab with more artistic talent than I did
actually tried to draw a sunset with a green flash as you might see it from the beach not far from our lab.
Thank you very much.
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Microscopy: Fluorescent Proteins (Roger Tsien)

406 Folder Collection
Pei-jyun Guan published on April 16, 2017
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