Let me start out by saying how grateful I am to be here and have the privilege of addressing this wonderful gathering of students.
So I thank the Countess Bettina and the council for the opportunity to do so.
Now, when I was awarded the Nobel Prize in 1997, the Nobel Foundation asked me, as I’m sure they do all other laureates,
to write a brief autobiographical account and explain who are the major influences in life who had got me to Stockholm.
And I’ll start out by giving you one such example.
So, from 1972 to 1974 I was working away very happily at the Pasteur Institute in Paris in the laboratory of Georges Cohen.
And I was using skills that I’d developed in protein chemistry to study enzymes
that were involved in making the amino acid methionine.
And then I did something very faithful.
I wanted to extend my skills in protein chemistry.
And so I went to Cambridge on a course that was held there around Easter in 1974.
And at this meeting, at the dinner, I met this remarkable man, Frederick Sanger.
So Fred is one of 3 scientists who won 2 Nobel Prizes for chemistry, the other ones being Madame Curie and John Bardeen.
And of course Linus Pauling won one for chemistry and one for peace.
Fred’s first Prize as you can see was in 1958.
He sequenced the first protein, the insulin.
And the importance of this was not just that it was the first protein,
but it demonstrated that proteins had a defined sequence which some people doubted.
Although Fred said that he didn’t.
But the important corollary to this was that if it had a defined sequence, that information had to be stored somewhere.
And then his second Prize came in 1980, awarded jointly to Wally Gilbert and Paul Berg.
And Fred’s contribution was that he’d developed methods for sequencing DNA.
Now, when I went to work with him in 1974,
he was just developing those methods and applying them to the genome of the bacteriophage phiX174.
And actually Elizabeth Blackburn also participated in that effort as a PhD student.
And what Fred asked me to do was to sequence the proteins that were encoded in the genes that he was sequencing
because he wanted to be sure about the accuracy of his DNA sequencing methods.
And this was a way of checking that.
In retrospect it sounds banal but of course there was a huge pay off.
And that was that we discovered triple overlapping genes in those bacteriophages.
That is to say that all 3 phases of DNA were being used to encode different proteins.
This was mentioned by Hamilton Smith yesterday.
I still find it a very remarkable observation.
And really this set the course for the rest of my scientific life up to the present day.
And so the first lesson I have for young scientists is you should choose an excellent mentor as I did in Fred.
And as I’ve said, the reason that he was interested in me was because I had these skills in protein chemistry.
And according to my friends, I developed these skills at a very early age.
This is me reading this book, “The ABC of Protein Chemistry”.
(laughter)
Now, the next thing that Fred decided to do was to move to a somewhat larger genome
that’s found in the mitochondria of human cells.
So this is a modern depiction of mitochondria.
Here is the nucleus of the cell.
And what you can see is that the mitochondria extend throughout the cytoplasm.
And sometimes they’re in this fused state where they form a reticulum.
And at other stages they’re all in this fission state for example during apoptosis.
And there is a dynamic equilibrium between these 2 states.
And so this is rather different than the text book picture.
At least the text books that I read of the mitochondrion.
So they’re all joined into this dynamic reticulum.
And within each of these cells there can be up to 3 to 400 copies of mitochondrial DNA.
In a haploid cell there would be one set of nuclear genes but several hundred copies of mitochondrial DNA.
So the genome was sequenced.
It’s about 17 kilobases of circular double stranded DNA.
And again my task was to try to identify the protein genes that were... the genes encoding proteins in that genome.
There are 13 such proteins.
So the mitochondria contain about 2,000 different proteins in human cells
and only 13 of them are encoded in the mitochondrial genome.
The others are the products of nuclear genes.
And they’re imported into the organelle.
And also within this genome there are 2 genes that encode RNase, 16S RNA, 12S RNA
that become incorporated into the mitochondrial ribosomes
because the mitochondria have their own ribosomes, their own system for replication, transcription and translation.
And all of the proteins that go to assemble with these RNA molecules are coming from nuclear genes.
And then the other genes within this genome are 22 genes for tRNase.
These are represented by these circles.
And there are a minimal set that would translate the products of the mitochondrial genome.
And so the question was: What are the proteins?
And nothing was known at the beginning.
What was obvious was that all of these proteins were extremely hydrophobic.
And so I started looking in the inner membranes of mitochondria
and identified this cluster of genes here as encoding sub units 1, 2 and 3 of cytochrome oxidase
that Hartmut Michel talked about earlier this morning.
And then these 2 genes here which encode components of the ATPase complex that I’m going to talk about in a moment.
And actually these were another example, strangely enough, of overlapping genes.
And also from these studies on these proteins came verification of the modified genetic code that’s found in human mitochondria.
And then the other genes shown in green and blue were identified by other people, largely by Giuseppe Attardi at Caltech.
Now people had looked at the inner membranes of mitochondria,
where all of these 13 proteins were, in the 1960s using electron microscopy.
And negatively stained... That would negatively stained.
And what they could see was that the membranes were covered in structures -one pointed to here- that they called lollypops.
So there was a head and then the head was held to the membrane by a stick.
And it was an American biochemist called Efraim Racker who demonstrated that these were the ATP synthesising complexes
that were involved in generating ATP within the mitochondrion.
Now, Henri Matisse also knew about mitochondria.
The lady who showed us around the exhibition the other day had a different story but these are obviously mitochondrial membranes.
And these are ATP synthase complexes.
And here is a different view of the mitochondrion, of these lollypop structures taken more recently by a group in Groningen.
And what one can see here is that these lollypop structures are arranged in pairs.
And then the pairs of structures make long rows.
And we now know that these rows extend around the edges of the cristae of the mitochondria.
And actually it’s these assemblages that give the inner membranes of the mitochondria their highly invaginated structure.
And if you remove the capability of these complexes to dimerise, you lose the crystal structure inside the mitochondria.
So what I did then for the next 20 odd years was
to build up a structure of this complex at molecular level largely by x-ray crystallography.
Of course we had to identify the protein sequence, all of the protein sequence,
the genes, crystallise different parts and solve their structures by x-ray crystallography.
And this picture is actually based on more than 30 high resolution structures that we’ve solved over the years.
This part is the catalytic part where the ATP is being made.
You can see this interface opening and closing.
It opens and then the change between opening and closure is being driven by the rotation of this central shaft.
So here is the inner membrane of the mitochondrion.
And there’s a voltage across this membrane, sometimes referred to as the proton motive force.
Minus 200 millivolts.
And that voltage drives that rotation.
I’ll explain it in greater detail.
This part here is referred to as the peripheral stalk.
And it’s linking the external surface of the catalytic domain to this part down here.
So all of this is static, it’s the stator relative to the blue rotor.
And you’ll notice that whilst we’ve got exquisite details for most of this structure in this region here
that detailed information is missing.
And that has been my quest now for several years, was to get that information.
Because this is a critical part of the enzyme
because it’s in this interface between the green protein and the rotating ring that the rotary torque is somehow generated.
So what the enzyme is doing is to convert the voltage across the membrane into this rotary motion.
And the rotary motion then makes the catalytic part bind ADP and phosphate and turn it into ATP.
And each of you today will make 60 kilograms of ATP in your mitochondria by this process.
So it’s a massive process.
This is an extraordinarily efficient machine.
I’ve slowed down the rotation because it’s actually rotating between, somewhere between 500 and 1,000 times per second.
So it’s spewing out huge amounts of ATP inside your mitochondria.
Now, the structural characterisation was our contribution
but there are also important bio-physical experiments done in Japan by Kinosita, Yoshida and Noji.
So guided by structures we’d done, they build this, first of all built this chimera.
So here’s the catalytic part, upside down relative to the way I’ve been showing it.
And here’s the central part of the rotor.
And so what they did was to attach an actin filament from mouse skeletal muscle and then added ATP.
Now the filament is big enough to be seen in a fluorescence microscope.
They made the filament fluorescent and so they could observe the rotation.
And this was direct proof of the rotary mechanism that we suggested on the basis of looking at our structures.
And then in more recent manifestations of this experiment.
They’ve replaced the actin filament with gold beads.
And look how... This is drawn to scale.
Look how big the bead is relative to this little biological machine
but yet it’s able to turn this bead around, speeds approaching 150 per second.
So this is one of their experiments here.
This is the actin filament going around like a propeller.
And that rotation depends upon the hydrolysis of ATP.
So here in this experiment ATP is the fuel.
And one is looking at the back-hydrolytic reaction.
And in the synthetic sense when the enzyme is making ATP, the rotation is in the opposite sense.
It’s driven by the voltage of -200 millivolts across the inner membrane.
And so what about the problem of how to solve the rest of this structure?
And you’ve heard many wonderful talks over the past few days describing various structures
most of which have been solved by growing crystals of those proteins
and solving the structures to atomic resolution by x-ray crystallography.
But there is another method which is emerging
and in the next year or 2 you’re going to see some spectacular atomic level structures
coming out from this procedure of cryo-electron microscopy.
Now you heard an explanation of it earlier.
But I’ll go over it again.
So here in this top panel here there are particles of purified ATPase complexes
that have been frozen in vitreous ice in a hole in an electron microscope grid made of carbon.
And there is sufficient contrast that you can see these particles.
So what you do is you pick them, as depicted in these panels here, and then classify them into these different groups here.
So these are different views, different aspects of the ATPase complex.
And this particular group...
That peripheral stalk is on the left.
In this group here it’s on the right.
And in this group here it’s hidden around the back.
And so from this information you can build up a 3 dimensional model.
And you can...
With appropriate quality of information you can get to atomic resolution.
And this is a principle of it.
So here is this 3 dimensional duck at the top.
And what you’re doing is you’re recording different 2 dimensional views which you then turn into 2D Fourier transforms.
And then from this you can transform that into a 3D Fourier transform of the object.
And then you transform it back again.
And you’ve restored... You’ve solved the structure of the original image.
And so we applied this structure to the ATPase complex from 2010 to 2012
in collaboration with a former student John Rubinstein who is in Toronto.
We got these pictures here at 17 angstroms resolution.
It sounds modest but actually there’s a lot of information in it.
The part in the middle of course we already knew the atomic structure and the green part down the middle.
But these blue parts here were new.
This was new information.
At modest resolution but it was new information.
But it was an awful lot of work.
It took 2 years and one had to pick 60,000 of these particles by hand
and assemble them into classes and solve this structure in this way.
But there have been recent advances in cryo-electron microscopy
so that it’s now possible to determine atomic resolutions of protein complexes that are greater than 200 kDa.
And actually this number is being driven down to lower molecular weights all the time.
And the fact that it’s made... the difference is in the development of new detectors for electrons
that you associate with the electron microscope.
They’ve got increased. So they detect the electrons directly whereas the previous detectors
that were used detected the electrons indirectly.
The electrons made light and you detected the light.
And this has meant there’s been a big increase in signal to noise and much faster acquisition of data
so that you can now collect the images on a movie.
And this... Steven Chu referred to this point in his experiments yesterday.
And so by collecting the images so quickly you reduce one of the problems that blurred out the images that you are seeing,
namely the impact of the electrons on the protein complexes made them move.
But if you collect the images quickly then that is reduced.
And of course you reduce any possibility of mechanical motions that are coming from the movement of the microscope itself.
And concomitant with this of course as usual has come big improvements in computational methods.
And so just a few weeks ago we collected 10,000 particles from an ATP synthase from a fungus.
It’s a thermophilic fungus called Pichia angusta.
And in 2 weeks from start to finish after purifying the enzyme
as on the left collecting the images as in the middle we’d computed this image now at superior resolution
that had taken us 2 years in the past.
There’s much more detail in this structure.
We’ve not actually looked inside to interpret it because we know that by collecting more particles
-and that’s going on right now- that we can get this to even higher resolution.
So this is a very exciting development in structural biology and it is undoubtedly going to have a huge impact.
Now another thing that we’ve done over the years was to compare the mitochondrial enzymes
on which almost all the structural work is being done with bacterial complexes.
There’s much less structural work on bacterial ATPase complexes.
But you can see there’s a great deal in common.
There’s a common core. The catalytic parts are very similar.
The rotor rings are similar. But you’ll see that they have different numbers of C proteins in the ring.
This is not a constant. And that has important consequences.
There is an A protein that is actually providing the pathway for protons
to get through the membrane and is involved in the generation of rotation.
And then there’s a peripheral stalk in both cases but their compositions differ.
And the biggest difference is that in the mitochondrial enzymes there are 6 additional proteins in the membrane domain
which have got nothing whatever to do with making ATP but they turn out to have great significances.
I’ll show you towards the end of this talk.
So this is the way we think rotation is generated.
Here’s the A protein that we lack...
where we lack atomic resolution data.
And the idea that came from Wolfgang Junge is that there’s a half channel
that allows protons to access a negatively charged carboxyl group.
So there’s a negatively charged carboxyl in each of the C proteins that make the ring.
And in this model there are 12 C proteins in the ring.
And here the carboxyl is negatively charged.
The proton goes in through the half channel, neutralises it, makes it more hydrophobic
and therefore simply through Brownian motion the neutralised carboxyl
will move to this position here bringing another negative charge.
Another proton will go in and so you can see that as this happens in this stepwise fashion,
the protons get carried around the ring until they reach this site here
where it’s imagined that the properties of this part of the interface makes the carboxyl re-ionise,
re-generate the negative charge and now release the proton on the other side of the membrane through another half channel.
Now if there were 12 C proteins in this ring to generate a 360 degree rotation, you’d require 12 protons.
And since this rotor is attached to the stalk that I showed you earlier, the blue stalk
which turns around in the catalytic part and since the catalytic part contains 3 catalytic sites,
every time the rotor goes round you make 3 ATP molecules up here.
And therefore the cost in this case of making an ATP molecule would be 12.
The symmetry of the ring divided by 3.
So that would be 4 protons per ATP.
So it’s the ring symmetry divided by 3 that gives you this cost which is very important and central to bioenergetics.
This cost of making ATP molecules.
Now to everybody’s surprise it’s turned out that the different ATPases from different species have different ring symmetries.
And we now know from our own work that the ATPases in metazoans from man down to sponges,
porifera, have rings with 8-fold symmetry.
Yeast has 10-fold symmetry.
And then eubacteria -has been demonstrated largely in Frankfurt- the eubacterial rings have anything from 10 to 15-fold symmetry.
And then spinach chloroplast where there’s an ATPase complex has 14-fold symmetry.
And so from this you can deduce that the cost, the number of protons per ATP is 2.7 in metazoans
and it goes up to 3.3 in yeast and then up to 5 protons per ATP in some eubacteria.
So what does this mean? So, if you look at the structures of these rotor rings.
So they’re drawn to the same scale in cross section.
This is our human ring that’s been driven round requiring 2.7 protons to make each ATP.
And this is the one for example found in the cyanobacterium synechococcus.
It’s got 15-fold and this organism pays 5 protons to make each ATP.
So it’s less efficient than the metazoan machine.
And why has this situation evolved.
Well, we think it’s because that in the metazoan mitochondria there is this very large membrane potential, minus 200 millivolts,
which can drive the ring whereas these organisms live in relatively impoverished energetic circumstances.
They generate a lower membrane potential.
And so the cost they have to pay then is to drive a bigger ring around in order to make 3 ATP molecules.
So this is a low gear in the machine and this is a high gear.
And this is the highest gear that is being observed.
I think it will be the highest gear that exists in biology.
So that’s one way in which ATPases differ between man and bacteria.
And the reason we’re interested to know this is we think that the ATPase complex
is actually a very good target for developing new antibiotics and new drugs.
And one of my students, Alice Zong is here in the audience.
And she has solved the structure of the enzyme from mycobacterium smegmatis
in order to be able to develop new drugs to combat tuberculosis.
Now, another way in which they differ, the bacterial and human enzymes, is in the way they’re regulated.
And I’ll just talk about the regulation of the mitochondrial enzymes.
But first I need to tell you that whilst, as you’re familiar, many proteins of course are structured,
they have alpha helices, beta sheets and so forth.
But there’s another class of proteins that has come to the fore in the past few years.
And these are intrinsically disordered proteins.
So you find them. And they have no secondary structure.
And they have common features.
They’re often heat-stable, have a large number of charge residues, a few bulky residues.
Many of them are involved in regulatory mechanisms.
And often they will bind to more than one partner.
That is to say they can form more than one structure depending upon which protein they’re interacting with.
And it turns out that the inhibitory region of the regulatory protein for the ATPase
-it’s called inhibitor of F1 ATPase, IF1- is intrinsically disordered.
And this protein is heat-stable. A large number of charge residues. A few bulky residues.
It’s predicted to be disordered and most importantly its nuclear magnetic resonance spectrum demonstrates
that indeed it is a disordered protein.
Now, this realisation was somewhat surprising because some years ago
we’d solved a structure of the inhibitor protein bound in the ATPase complex.
So this is the blue rotor in the middle here.
And this is an interface, one of the 3 catalytic interfaces.
And this alpha helix you can see here has inserted itself in this interface and has stopped the machine dead.
So it’s rather like sticking a stick in a rotating bicycle wheel.
You put the stick in, rotation stops.
And that’s what happens with the ATPase.
And in order to form this inhibitor complex you have to hydrolyse 2 ATP molecules.
So the idea was or is that under conditions of anoxia, for example during an ischemic heart attack,
that the proton motive force would become dissipated because of the onset of glycolysis.
This would then make the enzyme to start to run backwards.
It would destroy the ATP that had been made.
But this protein becomes activated by a mechanism. I won’t describe.
It’s normally quiescent.
And then intervenes and stops the back reaction.
And then if you restore the proton motive force, put it back under conditions
where there’s oxygen, the rotation goes in the opposite sense and the inhibitor is removed.
So this has remarkable property from an enzyme point of view of being a unidirectional inhibitor.
It only inhibits ATP hydrolysis and not synthesis.
Now what we’ve just done is we’ve managed to solve structures where we had 3 inhibitor proteins bound to the complex.
We had very large excesses of inhibitors with specific mutations that I won’t describe.
And what we could see...
So if you imagine that the inhibitor protein is completely disordered in free solution,
it then starts to bind to the ATPase complex through the most open of those 3 catalytic interfaces,
through the so called empty E interface.
And then this little nucleus of an alpha helix starts to form.
The rotor then rotates through 120 degree step driven by the hydrolysis of the first ATP. The site closes.
You can see that the aspect that you’re looking at, that the rotor has changed.
And now the helix can elongate itself and forms a somewhat longer helix.
And then there’s another 120 degree step, the rotation of the rotor driven by hydrolysis of the second ATP.
And now the inhibitor can extend its structure to the one that we’d observed in the past.
So this is a very nice observation which was unexpected in many people’s minds
that one would be able to observe folding intermediates of intrinsically inhibitor proteins by x-ray crystallography.
And this is... This paper was accepted yesterday by the PNAS.
Now I want to tell you about another extremely important way of uncoupling the ATPase.
And this is via something called the permeability transition pore.
So the phenomenon of the permeability transition has been known for more than 30 years.
But there was no molecular explanation. So let me explain.
So on the left... Don’t bother about the left.
This is a depiction of apoptosis which is familiar to you leading to cell death.
On the right is a depiction of how people think necrosis, another form of cell death, is triggered.
So the idea is that calcium is released in response to various stresses.
It could be reactive oxygen.
It could be a variety of things.
So oxygen in this case... calcium ions are being released from the endoplasmic reticulum into the cytoplasm.
And under those conditions the calcium ions are rapidly taken up by proteins
in the inner membranes of mitochondria into the matrix of the mitochondria.
And this influx of large amounts of calcium has the effect of opening a pore
which is called the mitochondrial permeability transition pore.
And it’s regulated by a protein that Kurt Wüthrich mentioned yesterday, cyclophilin D to which cyclosporine A binds.
So cyclosporine A actually prevents the opening of this channel.
And then what happens is that this channel, which is a big, big channel, it’s said to release molecules up to 1.5 kDa.
This disgorges the contents of the matrix of the mitochondria.
Water rushes in. The mitochondria swell and then they burst.
And then this triggers this cell death process by necrosis.
So that those phenomena are well studied and well known.
And the question has been: What is the mitochondria permeability transition pore?
So you can look at this review in Nature 2 or 3 years ago
and what it tells you is that the permeability transition pore is made of a dimer of a transport protein.
It’s called that adenine nucleotide transporter.
What it’s doing is to remove the ATP made in the matrix
and take it outside to make it available to the rest of the cell and bring back ADP.
So according to this model the channel is composed of 2 of those proteins and 2 other proteins in the outer membrane
that are called the voltage dependent anion channel.
And this is completely wrong.
Because the permeability transition pore actually resides within the membrane domain of the ATPase complex.
So what we’ve done over the past 18 months using RNAi in human cells,
is to eradicate each of the proteins in the peripheral stalk, each of these proteins,
the C proteins then by another means, I won’t explain, the A proteins and also the gamma proteins.
We’ve done each of these one at a time and looked to see what was the impact on the opening of the permeability transition pore.
Removing this protein had no effect, this had no effect, this had no effect, this had no effect, no effect, no effect.
But the ones that were important were e, f, g, DAPIT 6.8 proteolipid and B.
And so these are the so called supernumerary proteins.
Each of them appears to have one transmembrane helix.
We don’t actually know how many copies of each there are.
But we’re determining that at the moment.
We don’t know which way round they are.
Is it N-terminus in or out. But anyway the suppression of any one of them abolishes this permeability transition.
But we’re still not able to say precisely what makes the pore
because when you suppress E you also lose G from the ATPase complex and vice versa.
And there are similar relationships between these 2 proteins.
So we can’t right now say exactly what it is.
And so our conclusions is that those proteins are involved and A and C are not.
And what we’ll do is now determine the precise structure of this pore.
We’ll find inhibitors which have got tremendous medical potential.
And so my pursuing this enzyme complex for now 35 years has provided some more lessons for young scientists.
You need to choose your topic well.
You’ll definitely need stamina if you’re going to solve a problem like this.
And of course everybody needs some luck.
And then you may eventually end up in Stockholm as we did in 1997.
So this is Paul Boyer, Jens Skou and myself, the chemistry recipients.
And if you look carefully, you’ll see here on the left is Steven Chu receiving his physics prize.
So thank you very much.
Applause