Now we come to the third lecture this morning, and now it will be given by Professor Roger Tsien
from the University of California, San Diego which is actually in La Jolla and he also,
he shared last years Nobel Prize for the discovery and development of GFP.
Now he will speak about another or related topic which is building and breeding molecules to spy on cells, tumours and organisms.
So please, Professor Tsien.
(applause).
PROFESSOR ROGER TSIEN. I too would like to thank Countess Bettina
and all the other organisers for their tremendous effort in putting together this meeting.
And today because the students and the post docs are the explicit audience
I thought I would try to bring in some suggestions of how you might start your careers and choose what to work on.
Based on my own experience for good or for worse
and I hope to show you some of the places we made mistakes as well as the places that we got lucky.
And so I’ll begin by, since this is a chemistry meeting,
by discussing creativity in chemistry which typically can be either an analysis or synthesis.
And also this is of course, this particular prize has a close relationship to biology
and so I should remind you that in mainstream biological research our questions are:
how do natural bio molecules and biological systems work?
And Marty Chalfie just gave you some beautiful examples of that sort of investigation.
In mainstream chemical research, particularly when I got started, when I was introduced to it,
the emphasis was very much heavily on the total synthesis of natural products,
which was essentially asking how do human beings artificially re-synthesis natural bio molecules
in ways that may or may not resemble the way the original organism made that.
And I guess I wasn’t quite happy with either one and that was part of me
and I’ll explain a little bit about why I’m such an ornery sort of person
and I wanted to combine the two in a different way which is synthesis but applied to biology,
can we design and build new molecules that will perform amusing, we hope and maybe useful functions in biology.
And this is sort of fun because it’s sort of like architecture or sculpture on the molecular scale.
And also is a little less competitive
because you may have several groups all racing to build the same natural product after it has been synthesised.
And the first person is the one who first achieved the total synthesis or you could have the people
who are first trying to clone the gene for this particular function and then it’s a competition.
But in architecture or sculpture you're not, if you’re especially moulding a piece of clay,
you're not competing with anybody, nobody else is going to make exactly the same thing you are.
And that made me feel a little better about, because I wasn’t,
I was doubtful that I was a very good competitor so I like to be able to have my own playpen to work in.
And yet it’s also a rigorous test of one’s understanding of molecular systems
because not just ability to make things but to predict their properties, that is to make something novel.
Of course you can always rationalise after the fact, oh I think I know how this molecule works.
Well you really think you have a better evidence that you understand it when you can make it ab-initio.
And when I started at these traditionally this was considered something only drug companies would have the resources
to do big industrial teams, academics didn’t really try to do this strange combination of chemistry and biology.
So why did I do it, well I have to admit that engineering runs in my family,
I just happen to come from lots of lots of engineers,
but I soon realised that biology has the most interesting grand questions in science that I felt were doable by individuals.
At that time of course I was, you know I saw that it was very interesting, what is the origin of the universe,
what are future sources of energy, what are, how does the, well anyway, how does nuclear physics work,
where do atomic particles come from.
And I felt and realised pretty quickly those need enormous teams of people.
So in order to work on an individual scale, biology still has some of those wonderful questions.
And Marty beautifully alluded to them.
And I personally have been fascinated since childhood by pretty colours so that’s me.
Finally as the youngest of three children I needed to find an ecological niche
which is distinct from parents and older siblings and there’s a whole book about this psychological urge,
if any of you are youngest children or if any of you are eldest children
who have to cope with bratty youngest children I suggest you read this book.
Which is all about how Charles Darwin was driven by the need as a youngest child,
the oldest child was going to inherit the family fortune, typically the second one went into the army,
the third went into the clergy, he was supposed to go into the clergy, he didn’t like it,
he much preferred collecting bugs and beetles.
And eventually to find something different to do he became a naturalist and the rest is history.
And finally I’ve been trying to avoid competition because I’m scared of it
and was useful to realise that interdisciplinary barriers prevented most biologists
from knowing how to manipulate molecules other than the traditional linear alphabetic strings,
twenty amino acids, four DNA right nucleotides, one extra for RNA.
Almost adds up to twenty six, almost, that’s what most biologists knew how to manipulate
and therefore anybody who could make molecules that weren’t just linear, that were more like pictorial diagrams,
in other words more traditional chemistry would have big opportunities and few competitors, at least back when I was starting.
And I urge you maybe to look for the same areas where interdisciplinary barriers create a niche that is thinly populated.
So as you’ll see my usual approach has been to identify some sort of need in biology
that I think is significant and preferably one whose solution would suit my neurosis
and I think you’ll see some of these neurosis that I have.
And then I identify what properties the molecular solution would need to have
and you know you cannot solve all the problems perfectly, you cannot make the perfect molecule the first time
so you have to have some idea what is the most relevant priorities to start with and what can be deferred to later.
Then you identify such a starting point and then you try to modify it
to gain as many of those that require other properties as possible.
And this is the first stage at which you need real experiments and some bravery.
So this is typically now in my lab where I have to recruit some member of my lab,
I have to convince somebody that this is a problem worth trying.
The first three I can do sitting there nowadays ever more at the computer instead of,
used to be I had to go to the library and we come up with some suggestion, some potential molecule that we want to make.
Finally we try to test it biologically and then we often have to bring in collaborators
who are experienced in that form of biology because unlike Marty I am not wedded to one model organism,
I have never been able to settle down in that sense.
And finally this whole process needs to be reiterated as we find out what's wrong with our existing molecules
and what do we most need to improve.
So I actually started with intercellular calcium as the first biological target, actually it was my second
but the first was so hard that I gave up on it which was membrane potential to begin with.
And I’ll just explain briefly why calcium was important but I think Erwin Neher already introduced this topic,
that fluctuations in free calcium are crucial for muscle contraction, neuronal communication
which is synapses or particularly what he discussed, sperm egg fertilisation,
which is another form of secretion, enzyme activation.
I believe he mentioned how fast and localised those signals can be and that the calcium levels that you’re looking at
are very low, on the order of 10 to the -8 to 10 to the -6 molar in the resting state,
possibly rising to 10 to the -5 or -4 at the very, very highest point, very locally.
But this is a tiny fraction of the calcium that you measure if you do traditional analytical chemistry
which involves as usual grinding up lots of cells and burning them up and measuring how much total calcium,
which gives you numbers much, much greater than 10 to the -3 molar.
Any damage to the cell membrane causes a flood of calcium to come in from the outside
and those levels are in us around 10 to the -3 and in marine creatures around 10 to the -2 molar.
So again these are huge numbers compared to what you want to measure.
And finally there’s a huge amount of magnesium that you must work in the presence of.
The previous calcium indicators aequorin was one of the very best and that’s due to the work of Professor Shimomura
but then various synthetic dyes had been made, all of them with certain drawbacks for looking at single cells in particular.
And so the way I started was trying to find something new.
I decided that the most important problem was the calcium to magnesium selectivity.
Rather than worrying immediately about how good a diet was, I just wanted that selectivity.
I figured that I could fix the dye part of it later.
So I started with chelator called EGTA which was the only then known chelator in common use
that had the five orders of magnitude selectivity for calcium over magnesium that was necessary.
But you see this molecule has no chromophore, it doesn’t absorb even decent amount of UV,
ultraviolet light let alone visible light, so how do you get it to tell you anything.
Well you would obviously have to add a chromophore, the simplest chromophore that is chemically stable is a benzene ring,
so how could you stick a benzene ring into this molecule.
Well one direct option is to stick a benzene ring down here.
On replacing this single bond between the two in the ethylenedioxy unit, by a benzene ring.
And I thought about it and realised this would be a little bit more difficult to make.
But then there was another alternative that you could put the benzene ring and insert it annulated basically into this side.
And for symmetry it would be easier to do it on both sides.
And this molecule will have the great advantage that now when calcium bound in here,
and this I did now with CPK models before computers, actually built the model,
tried to put calcium in here and saw that the nitrogen to carbon bond would have to twist upon complexation with calcium.
And that nitrogen carbon bond twisting would disconnect the nitrogen from the rest of the benzene ring
and cause a spectroscopic change.
And also the presence of the benzene ring would lower the PKA of the nitrogen so that it wasn’t so prone to protonate
and that would make the calcium indicator PH insensitive.
And so this is the molecule I chose to make and fortunately it took only about a month to do so.
Now of course any good synthetic chemist would be able to now do it in six hours or so,
would look at it and know how to do it but I was really not a very good chemist
but I’m glad it didn’t take more than a month
because I was already at the last stages of my PhD having basically ruined three other projects,
this was my last desperate attempt to get something that would be a road for the future.
And fortunately it didn’t take that long, in a month I had the proof that I could make this
and it did indeed have the properties of very high calcium to magnesium selectivity.
A big change in the UV spectrum, fast kinetics and no PH dependence.
Exactly as designed, it was a lousy chromophore because it was only a benzene ring but that was expected
because that was part of the principle.
And then it took about another eight years I’m afraid, through many evolutions to turn it into fura-2,
I think is one of the molecules that Erwin Neher might actually have eluded to,
where obviously we have now extended the chromophore a great deal and now this has a useful green florescence.
But then came the problem of how to get this inside the cell and with all of these negative charges,
this is fura-2 again, it has five negative charges, four of them are essential for binding the calcium up here,
one of them is down here, partly for synthetic convenience and partly so that we don’t have a detergent.
If this was non polar this entire zone of the molecule could be relatively greasy and that might make a detergent.
So we’ve spoiled the hydrophobicity by this negative charge here.
But with all these negative charges this molecule won’t cross plasm the cell membrane very readily
and I too was obsessed with the problem that Marty has eluded to, that we want to work on live cells,
we don’t want to have to kill cells and fix them and permeabilise them for everything we want to work live.
And the traditional answer would be to micro inject this material inside cells.
Eventually Professor Neher’s discovery of the patch clamp made this much easier,
but at the time this didn’t exist and I was really bad at doing micro injection,
I didn’t have the patience nor the manual dexterity.
So here’s an example of compensating for my neurosis, I had to develop a chemical solution
and that was to discover a protecting group in organic chemical nomenclature which makes an ester that is hydrophobic.
Blocks all the negative charges but there’s lots of those hydrophobic, you know ester groups.
But the key is that this acetoxymethyl ester is one that can be de-protected by the cytosol itself.
In other words our cells contain enzymes that break this up
and spontaneously revert with the help of this enzyme back to all the negative charges.
And so simply by making this and putting it outside self, it will soak in and regenerate the material
and hey we have a chemical solution to the need for micro injection.
When calcium binds after that, it twists this nitrogen carbon bond as I’ve tried to draw crudely here
and that shifts the spectrum.
So one very simple example, I won’t pick the beautiful one from neurons that may have already been shown,
this is the beginning of life for sea urchin eggs so it’s sort of cute.
Here is a sea urchin egg that has just had the die put in it, sperm was just put in the dish
but the sperm haven’t quite found the egg yet.
So this is time after addition of sperm into the dish.
But by thirty six seconds one sperm clearly has made it and has touched the egg here
and started this wave of high calcium which is symbolised here by the warm colours.
And over the next eight seconds it gets a quarter way, fifty two seconds it’s half way and by sixty eight seconds
which is less than a minute since we started, the entire egg is fused with this high calcium
which we pick to be this bright red colour because I wanted to pick pretty colours
and I psychologically associated blue with rest and red with activation.
That’s entirely a human choice, it’s quite common now in the literature
and I believe I may be one of the people to blame for that,
but it enabled my colleague Rick Steinhardt to call this the blush of conception.
Then afterward because high calcium maintained is toxic to cells,
this calcium wave has done many things like trigger all the subsequent events of development,
at least early development and caused a secretion of a protective membrane around the egg,
a sort of chastity belt because the egg having had one sperm, definitely does not want any more sperm.
And so it raises this barrier to protect itself from any additional fathers.
And so eventually the cell cools back down to a low calcium.
Now that was all well and good and it fed my career for a while but I then wanted to extend myself beyond inorganic ions
and the problem is, is that calcium is nice, it’s spherical but the next most important messenger is cyclic AMP.
The actual prototype of the concept of an internal signal transduction molecule
whose importance was recognised by Nobel Prizes to Sutherland for the discovery of the messenger
and then Krebs and Fischer for the mechanism by which it activated cell function.
But for me the problem was that as working on this mixture of chemistry in physiology and calcium being an inorganic ion,
I was consigned to the so called salt and water gulag at Berkley
because there was going to be a big reorganisation of the biological sciences
and people who worked just on salt and water were very sort of low class citizens
compared to the people who worked on molecular biology and worms and proteins and glorious things like that.
And so in order to make myself look respectable I wanted to be able to work on this molecule, this famous messenger.
But here too, just as before with the calcium the existing means for measuring cyclic AMP were
and still mostly are destructive, people even now still grind up millions of cells
and buy a kit from Amersham or something to do radio immunoassays after the cells are all dead.
And therefore the dynamics of intercellular, cyclic AMP were obscure.
And there’s another problem that there’s all these micro molar levels of this messenger to be detected in the presence,
again swamping amounts of other adenine nucleotides, like ATP which is what cyclic AMP is made from, is present,
that’s maybe five millimolar where the cyclic AMP is less than a micro molar.
So again we have a huge selectivity problem
and again the solution may be hijack somebody else’s already existing cyclic AMP sensor of which there are several possibilities.
But then you would have to label them with fluorophores to detect the cyclic AMP conformational change.
And so what we came up with is this scheme for hijacking this cyclic AMP dependent protein kinase
which is again what Krebs particularly had gotten the prize, his prize for.
It consists of two regulatory sub units, two catalytic sub units and when cyclic AMP binds to the regulatory sub units
and the cyclic AMP are symbolised by the yellow circle,
then the regulatory sub unit undergoes some sort of conformational change,
kicks out the catalytic sub unit which is set free to do phosphorylation.
But in the process it splits the protein sub units apart.
And then I realised that we could use the trick called florescence resonance energy transfer or FRET
to monitor this process in situ, in a living cell, if only we could put say fluorescein on the catalytic sub unit,
rhodamine on the regulatory sub unit and in this complex we would get FRET because the fluorescein and rhodamine would be close.
And just like with aequorin and GFP when they are close to each other the blue light from aequorin is,
the energy is transferred to the GFP which then glows green, here it’s a green red example but the same principle applies.
On the other hand there you would get FRET, blue light into the fluorescein,
instead of giving you green out would give you red out.
And then when cyclic AMP split the sub units,
blue light into the fluorescein would just simply give you green because the rhodamine was too far away.
And we got that to work and I won’t take the time to show you all the examples, but it did require that we express
and purify these sub units in E-coli and I had to even move to San Diego to collaborate with Susan Tailor who had that working.
And it took my people a year sitting next to hers and learning from her how to handle the enzyme
in order to figure out how to label with rhodamine and fluorescein, without destroying the protein function
and to put it back together and to micro inject it into living cells.
And I kept telling you I hated micro injection and I still hate it but at least at this stage I was a full professor
and I could tell my students you learn how to micro inject.
I’m no good at it but you learn.
But clearly this was still a very cumbersome procedure that would be hard to generalise to other proteins
and so we wanted a general means to fluorescently label genetically designated proteins in living colours
and I needed two colours because I needed the equivalent of fluorescein and rhodamine and that’s the other reason,
the scientific reason aside from my personal obsession with pretty colours, on why we had to immediately,
once we got GFP, why we had to change its colour.
So indeed here, this is my take on the Douglas Prasher, I was delighted, I hadn’t heard Paul Brehm
but I knew from aequorin and Professor Shimomura’s work that there was this nasty component
that you had to get rid of called green florescent protein.
And then I realised that GFP if it had been cloned would be useful.
I looked this up in Medline and I was lucky that this was about a month after, this by coincidence a month after Prasher
and Eckenrode had published their paper and this is a copy of the paper.
By the way this is really in evidence for the importance of basic research and the blindness of some granting agencies,
this work, Douglas had tried to get the money from NIH, the National Institutes of Health, he was turned down.
The National Science Foundation which would more normally do marine organisms, they turned him down,
only the American Cancer Society of all things, which is actually rather translational,
had the courage to give him a very small grant which funded the earlier part of his work.
But poor Douglas did not manage in the early stage of his work to get something full length enough to work in E-coli
and so that led eventually to his departure from science.
Nevertheless the rest I think you know from Marty’s beautiful story, that Marty actually also got the gene
and was the first to show that it would express E-coli in worms by itself.
But let me just come back to my part of the story now, which is that actually wild type GFP,
despite its inherent advantages had some real problems, it had its main excitation peak in the UV
and only a minor excitation at the desired blue light wavelength.
And people didn’t, don’t really like to use UV light if they don’t have to.
And it brought excitation spectrum, already was enough to prevent it from being useful as a FRET acceptor
so we needed to fix this and its folding efficiency and codon usage and slow formation.
So here’s the excitation spectrum of wild type GFP, shown in the dotted line, there’s the big UV peak and the small visible peak.
And I had this strange hypothesis, I mean I had to speculate chemically what’s going on here
and I thought that maybe this chromophore as drawn,
this is the Shimomura structure that he proposed for the chromophore even before the cloning,
I thought maybe that’s responsible for this big peak.
And that maybe a small fraction dehydrates this bond here, putting an extra double bond, creating a sort of dehydroalanine unit.
That extra double bond might lengthen the conjugation and might be responsible for this little peak.
And in that case our job was to promote dehydration of serine 65 or what's left over.
So there’s a simple way to test this hypothesis, I asked Roger Heim
who was the post doc who could do the molecular biology, change this, what used to be a serine into an alanine.
With an alanine we now have no OH, we just have hydrogen and that you couldn’t do an elimination on.
So that should entirely drive the spectrum over to this peak.
So Heim did it, he made S65A and he proved that I was completely 100% wrong, completely the opposite happened,
instead of emphasising this peak it went away and all we got was the longer peak.
Well ok so you know nature tells you, dumb, dumb, start again.
So we then kept trying the other amino acids such as cysteine,
cysteine with an SH here should eliminate more readily than hydrogen sulphide, well ok sure enough it also emphasised this peak.
And then we went to threonine which only adds a methyl group here and that should eliminate just fine
but if anything that killed this, also killed this peak.
And so it happened to be about ten nanometres further red shifted
and the threonine is what every molecular biologist considers the most palatable substitute for serine, the most conservative.
So for that psychological reason we advocated serine 65 to threonine as the most suitable mutation for future use
because it now was six fold brighter and had gotten rid of this peak and was not suitable for FRET and so on.
And it also made expression a little easier and so on.
So that was all well and good.
So all of that was done without the help of a three dimensional structure and we wanted to get one
and when Jim Remington came to me and said let’s, would you like to collaborate on a crystal structure, I said great,
but we know there’s five other groups competing with us, they’re trying to solve the crystal structure too.
And that's a horse race, I hate these competitions and you see this is a constant theme.
How do we get ourselves out of the horse race and yet sort of still in it.
Let’s solve S65T, nobody is working on that one and that way we have it to ourselves
and even if somebody else scoops us on the wild type, we can still publish.
And we’ll publish it because it will be interesting, how did one extra carbon atom
and one methylene going from serine to threonine in one amino acid, improve the spectrum so much.
Well it turned out that Mats Ormo was one of the first if not the first to get crystals and to solve the structure
and that revealed this famous eleven stranded beta barrel with the chromophore hidden inside.
And the fact that the chromophore is so well buried explains in a sort of way why no other enzyme could be involved
because no other enzyme could ever get at it.
So we were very pleased with ourselves, we even made a yellow version by rational mutagenesis, semi rational mutagenesis
based on the crystal structure.
And then we tried to get it published in Science and we too had our problems.
The first referee said that it was, competent crystal structure, uninteresting protein, yeah,
not particularly interesting enough to be publishable in Science.
Second referee, very important protein, but it doesn’t,
this paper on the x-ray structure and mutagenesis does not answer the crucial question about GFP,
which is what is its function for the jelly fish.
(laughter).
What, how the hell is a crystal structure supposed
to tell you ever what the ecological function is in its native environment, it’s the wrong sort of technique.
But with two negative reviews they obviously couldn’t accept it.
I appealed and the most that the editor could do, even trying to be kind was to say well I’ll send it to a third referee.
And so that went out and the third referee never answered, never answered, never answered.
Months went by and then finally on the internet appeared a comment from a young lady called Fan Yang in George Phillips lab
saying hey everybody we just saw the crystal structure GFP and it’s coming out next month in Nature Biotechnology.
So we simply took that comment and emailed it to Science and next day
our paper was accepted without waiting for referee number three.
(laughter and applause)
So there’s a couple of morals of the story here.
But I have to say the internet is great but be careful about what you say on it
because you know had that comment not appeared we would have been way, way trailing in the dust.
So eventually we managed to make FRET indicators out of all sorts of biochemical processes like proteases,
eventually we got cyclic AMP working, though that was one of the more difficult ones actually,
calcium and phosphorylation indicators that measure protein phosphorylation and kinases
and so with this a lot of the dreams that I had came to pass.
But one thing also bothered me, we could never get an honest to goodness red
and that was always my favourite colour as a kid, bright red and we never managed to make GFP turn red.
And so we were both amazed and a little envious when a Russian group showed the corals which are not bioluminescent,
actually generate red fluorescent proteins.
So this came out of left field, we didn’t do it, another one I wish I had done.
But what they had not done was solve the structure of the chromophore, they just had a DNA sequence,
it didn’t explain why was this gene that was homologous to GFP turn red instead of green.
And that we were pleased to be able to contribute because they made the gene available,
fortunately we bought a copy and made it in bacteria and by a combination of mass spec and chromo peptide analysis
and some chemical tests and so on, we realised that where the chromophore GFP is this hydroxybenzylidene-imidazolinone
but stops here in its conjugation, what the red protein has figured out how to do is dehydrogenate this carbon nitrogen bond
and extend it by two units and therefore get to a long wavelength.
And this is a famous photograph of where we got the 2004 with a wide variety of colours.
But in the last few minutes I want to tell you that fluorescent proteins as good as they are,
have some major limitations, sometimes they’re just too big
and there are times when fusing GFP to your favourite protein does screw it up, alas.
The excitation wavelengths of 600 that up to now have not exceeded 600 nanometres
and that does not penetrate far enough through relatively opaque organisms like mice.
And then we are even bigger and more opaque and for that we need imaging techniques such as magnetic resonance.
And finally the great virtue of GFP and all the fluorescent proteins is that they are genetically transferable
but that is their Achilles heel when it comes to application to human beings
where both genetic therapy is technically difficult and morally fraught with some concerns.
So to answer both of those questions one of the areas we’ve been working on are infrared fluorescent proteins
and here we’ve had to venture completely away from the original eleven stranded beta barrel
and go to a completely new family of proteins, derived from the phytochrome series
and they use biliverdin a tetrapyrrol like chromophore
who a little bit of a tetrapyrrol was introduced in Professor Shimomura’s lecture as one of the luminescent luciferase
but a different tetrapyrrol that you and I already make in large quantity every day called biliverdin.
This is the first break down product of Heim, everyone of you is making about a quarter gram of biliverdin per day.
This can be incorporated into a protein that makes it florescent, makes a covalent atom that is florescent.
And now with these longer wavelengths we can much more readily see into an intact mouse.
This is the liver of a live mouse that has been adenovirally transfected.
Whereas previous florescent proteins were much, much dimmer, were hardly visible,
GFP was quite useless in this context because the liver is so darkly red as you know.
But finally we’ve been getting away from, back to my roots in synthetic chemistry,
completely getting away from florescent and genetically encoded ones for the use of clinical application.
Our target has been these proteases such as matrix metalloproteinase 2 and 9
that are so famous for their involvement in tumour metastasis that we could just steal a cartoon from a commercial website
so it’s very valuable to be able to see the activity of these proteases.
And the molecular scheme by which we do so is now quite different,
instead of FRET we are using something called a cell penetrating peptide
which is a polycationic peptide that has a bunch of positive charges, something like 6 to 10 positive charges.
And if that is fused to a payload that normally wouldn't be able to enter the cell,
that combination will stick to the outside of the cell
by electrostatic attraction of the plus charges for the outside, negative charges.
And then some of this gets endocytosed into internal organelles
and from there a little bit of it may be able to escape into the cytosonal nucleus, though there’s also some indication
that under some circumstances may be cell penetrating peptides can bypass the endosomal process.
But this as I’ve just described to you is a very indiscriminate process in cells and in order to make it selective
and thereby workable inside a living organism we had to figure out a way to stop it from happening.
And the way we do it is to fuse in a row of negative charges of matching but opposite charge
and that polyanion keeps the polycatine neutralised and busy and therefore not sticky.
As long as those two are held together covalently a cleavable linker.
I said it’s a cleavable linker because we want to be able to cut it apart and when that gets cut apart by something like MMP2
and 9 that the tumour has, a normal tissue does not have much of,
that locally releases the polycatine from its inhibitory constraint and then the polycatine can do its usual thing,
as if it had been locally injected at the site of enzyme activity, note that this is an amplifying mechanism
because one enzyme can cut many molecules and trigger the entry or at least cell adhesion of a large number of payload molecules.
So a little example of how this works is in these artificial tumours which are in xenograpts, this is the peptide,
in red is the acidic domain, lower case indicates d-amino acids, upper case,
in this case PLGLAG is a good substrate from MMP2 and 9.
Lower case arginines, these are d-amino acids and the red dye, Cy5, deep red so that it can penetrate flesh,
it’s florescent, is there and this was injected in to the animal systemically
and yet it homes in or is retained in the tumour when the normal tissues don’t hold on to this material,
so that the tumour lights up.
If you use a control peptide where you simply invert the stereochemistry of the four amino acids, PLL and A,
now they’re d-amino acids, the enzyme doesn’t recognise it,
even though the molecule is the same molecular weight in hydrophobicity.
And then the tumour does not light up with our dye.
This tumour happened to have been marked with GFP, it’s too bad human tumours don’t come with GFP already in them.
So finally to show you an example of how this might actually help surgeons, I have this little movie here
and I hope that the lights could come down for this one movie please, could someone please help me there.
And what you’ll see is this is the ordinary view and then the florescent view
and finally this is an overlay where we have combined the florescence on top of the regular view
and the florescence which is actually deep red is false colour green.
Now the green is not from GFP, I have to keep emphasising this,
reporters who don’t understand science look at this and say oh that’s Dr. Tsien putting GFP into the tumour,
this is a false colouring, we only pick green because normally there is nothing green inside a mouse, ok.
So this is an overlay that clearly distinguishes the florescence from the rest.
And so we move it around a bit so you can see that this is live and then in a moment there’s another view,
there’s the tumour and finally this helps the surgeon cut more accurately.
You can’t tell from one of the views where you just see the reddish flesh
but with the green overlay it becomes quite simple to go and snip out the tumour.
You want to get rid of everything green.
Now there’s one thing that the surgeons must not cut which is the nerve and I’m going to freeze it there
because we have a separate peptide that labels the nerve and there its florescence we encode as light blue,
not because it really is light blue, because again light blue is a colour that doesn’t normally exist inside flesh.
And this particular branch that’s coming out here is one that you wouldn’t have been able to see otherwise.
The rest is all obvious and anyone probably could have seen that there was a nerve there
or this trunk of white flesh but this hidden branch would be otherwise not so obvious.
As shown, there’s the florescence but if I stop it there you can see again the nerve is very visible
but that extra branch wasn’t visible except by the help of this molecular staining in the live organism.
And so here the job of the surgeon is to cut out the green stuff without damaging the blue material
and we think that this may eventually be a way in which all this molecular understanding and the proteases and so on
can help surgeons go on and make more accurate tumour resection
which when it works is still the quickest way and cheapest way to achieve a complete cure.
Ok so we can actually improve survival when we use surgery guided by florescence compared to traditional surgery,
this is time in weeks after surgery and in a very aggressive tumour
where only one out of twelve animals would survive otherwise,
we can raise it to almost five or six out of twelve
and so we think that this may be a form of personalised medicine where the imaging guides the therapy.
So my last is to comment some lessons for young scientists, I hope to have given you my feeling
which is try to find important problems but also to put your own neurosis to constructive use,
try to find projects that give you some essential pleasure, in my case the love of pretty colours.
Accept that your batting average will be low but hopefully not quite zero.
That your best papers may be rejected from the fashionable journals or when they finally do accept it for the wrong reasons
and grant proposals are just the same or worse for funding.
Learn to make lemonade from lemons, so often you turn out to be completely wrong but don’t just give up,
let nature teach you a lesson, sometimes persistence pays off.
I really want to emphasise prizes are ultimately a matter of luck, don’t be motivated or impressed by them.
And I’m not any smarter on October 9th than I was on October 7th, even though I’m a lot more famous by October 9th and so on.
Of course we have to find the right collaborators and exploit them kindly in a mutually beneficial way so that everybody,
you know is happy and gains from.
And in particular these are my collaborators and in a lot of, I have to really emphasise many a senior scientist get up
and say all the work, I didn’t do it, the work was done by the people in my lab
and that is not only true in my case, I couldn’t even have done the work
because I am personally incompetent in the techniques of molecular biology,
I have to confess I never learned how to run a gel or a PCR reaction.
I have yet to do one in my life.
And so really dependent on smart people who are willing to do all of this.
When I do have fun I do try to go back and make some dyes, that’s about all I know how to do and I don’t even do it very well.
It’s only between Christmas and New Years that the emails will let up enough for me to do it
and basically I sort of do a rotation in my own lab and generally come up with the opinion that if had to hire myself
I wouldn't do it, I’m so terrible at it.
(laughter).
Nevertheless we’ve had some fun and we draw pretty pictures such as this sun set with green flash,
drawn with the bacteria expressing all the different colours, so thank you.
(applause).