So good morning everybody and thank you very much for the introduction.
And I start off showing you a photo of where I work, that’s the Max Planck Institute of Biophysics in Frankfurt, Germany.
And there are about 200 people and they all work on membrane proteins.
So there must be something of special importance for membrane proteins.
Also Frankfurt University which surrounds us has a focus on membrane proteins.
And if you have a look at a cell of a higher organism, you see, all you see is membranes.
The outer membrane you see here, organelles, that’s the lysosome.
You see here the endoplasmatic reticulum, the mitochondria, the Golgi apparatus and you see the nuclear envelope membrane.
And what’s special for membrane is that they are electrically tight, they don’t allow it to...
They are impermeable for ions and other polar substances.
And as a result of that you can’t build up electric voltages across membranes.
And membranes are a barrier.
Therefore you have to get information across the membrane and you have to get substance across the membrane.
So these are wider functions and without membranes there would be no life.
And the membrane proteins are the active components of biological membranes.
And of course they have to catalyse the transport of substances across the membrane.
Maybe proteins, maybe ions or maybe nutrients.
And you have to get rid of the catabolites and you have to export proteins.
You have to get the information across the membrane and you have centres, normally called signal receptors,
that transduce the signal, amplify it across the membrane and in addition you have biological electron transfer.
That’s not so straight forward to see.
But it’s the case that that’s a very important membrane process for the synthesis and cellular respiration.
And some enzymes... Some membrane proteins are enzymes.
In particular, when the substrate is hydrophobic.
Also a virus... When a virus enters the cells first has to interact with membrane proteins.
And it’s of course of particular importance that when you have a drug most likely it will act on a membrane protein.
I go further and I say that most diseases are caused
by a dysfunction, understimulation or overstimulation of a certain membrane protein.
Also the specificity of signalling and interaction comes in at the membrane level.
But also at the transcription level because this decides which membrane protein is made in a particular cell.
And there are more than 1000 membrane bound signalling receptors.
You have heard about the GPCRs, G-protein coupled receptors, by Brian Kobilka.
And there are more than 800 of this type in the cell.
But the number of signal transduction chains leading from the receptor to the nucleus or somewhere else is very limited.
So when you interfere pharmacologically at later stages of signal transduction, you have the danger of undesired side reactions.
So the best thing is to interact at the membrane protein level.
We want to have the atomic structures.
We want to know the position of each atom of a membrane protein
in order to understand the mechanism of action as basic scientists.
If you are interested in curing diseases, you go for rational drug design.
That’s what also pharmaceutical industry does.
And the similar technique is virtual screening, where you ask the computer,
give him chemical library and the computer tells you which of the compounds might bind to a pocket of the protein.
Membrane proteins are important, have been recognised by the Nobel Committees
and probably the first Nobel Prize for membrane protein was given to Otto Warburg,
Prize of Medicine for the work on the respiratory enzyme.
Then Hans Deisenhofer, Robert Huber and myself got the Prize for the first atomic structure determination of a membrane protein.
It was a reaction centre of photosynthesis.
1996 Paul Boyer, John Walker and Jens Skou got the Prize for the work on ATPases
and you will hear about the work about the ATPases by John Walker later.
And 2003 Peter Arge and Roderick McKinnon got the Prize for their work on channels.
And 2012 you got Brian Kobilka for his work on G-protein coupled receptors.
I think that is not the last Nobel Prize for work on membrane proteins.
And when I started off, it was in the most frequently used text book, there was a statement:
“It is impossible to crystallise membrane proteins”.
And I belong to the persons who are challenged if somebody tells them it’s impossible.
So I started to think how to get membrane proteins crystal based on extended observation.
And in order to tell you this in some detail, I show you here a schematic drawing of a membrane.
You have here the membrane proteins.
And you have here the lipids.
Lipids form this electrically tight layer here and you have the membrane protein incorporated.
And what the difficulties causes with the membrane proteins that they have a hydrophobic surface here
where they are in contact with a side chain of the lipids whereas they are polar where they are in contact with the phases.
And as a result of that you have to use detergents or expensive soaps and you add them in excess and you solubilise the membrane.
The membrane...
The detergent micelles looking like this.
Displace the lipids and then you get a structure like this which is the membrane protein within a detergent micelle.
Also the lipids are taken up by detergent micelles.
And that’s the situation where you start to purify your membrane protein and you try to get out its function and its mechanism.
When you think on how to order a membrane protein with these properties in the form of a crystal you have two solutions.
You can try to form 2-dimensional crystals in the plain of the membrane and you can form then ordered stacks.
This is what I always considered to be most important.
I call this Type 1.
The more easily accessible alternative is when you try to crystallise the membrane protein within the detergent micelle.
And you will rely on polar contacts between the polar surfaces of the various membrane proteins.
So it’s the membrane protein which forms the crystal lattice.
And the detergent micelle actually more or less passively still surrounds the membrane protein’s hydrophobic surface.
And you have to make sure that you don’t get...
that the detergent micelle doesn’t get too big and you get some repulsion so that you cannot form a real crystal lattice.
So looking for the right detergent is the most important step in trying to get membrane protein crystals.
Most important you have to find a project which suits for your ideas.
And after some attempts I ended up with a cell called Rhodopseudomonas viridis.
That’s a purple bacterium.
And now they changed the name and so it’s no longer called... It’s now called Plastoglogus viridis.
And it’s full of membrane stacks and these membrane stacks are full of photosynthetic proteins.
So it’s rather easy to get substantial amounts which you need for crystallisation.
And it’s also rather easy then to purify them and you get colour changes
if your membrane protein doesn’t like the conditions where you work with it.
So after rather short time I got this kind of crystals.
And looking nice and also surprisingly they diffracted well from the very beginning.
And I have to say something about technology development.
It took about 20 hours to record that photograph using photographic films.
Nowadays you go to the synchroton and the recording of that photograph takes 5 seconds, maybe even shorter.
And to collect one data set it took me 3 ~ 4 months.
Nowadays it takes less than a minute to collect the data set.
That is really a change and speeds everything up.
But the bottleneck which we still have is getting the crystals.
So we succeeded, you see here.
You see here the structure of a photosynthetic reaction centre.
That’s the part of the protein complex which is incorporated into the membrane.
You have here the photosynthetic pigments here.
You have here electron supply to the primary electron donor here.
And you have here more or less a scaffold protein which may be important for the assembly of the reaction centre.
If you look for a space filling model, you see here in white you see carbon atoms.
And most of the atoms approaching the surface here are actually carbon, are carbon atoms.
You see here a row of blue atoms, nitrogens.
And people being used to look at structures they easily can see that actually it is an arginine residue.
There is a histidine residue.
There’s another lysine residue.
And so here we have a nice row of positively charged amino acid residues
which can interact with the phosphates of the lipids and position the membrane protein in the vertical direction
which is also an important job.
The photosynthetic pigments are shown here.
You see here the primary electron donor which is constituted of two chlorophylls
and then you have an electron transfer from here
onto that, onto that, onto that and then parallel to the membrane from here to here.
The entire structure is symmetric which was a big surprise, was unexpected.
And now people could determine more membrane...
more structures of photosystems, that’s the plant photosystem I.
And what I showed you in the bacteria reactions is simply that central part.
Then you have your many, many light-harvesting complexes made of chlorophylls.
These are inner light-harvesting complexes and here you have outer light-harvesting complexes.
But the core of the reaction centre, of the core of the photosystem is the same.
And this is a comparison of the bacterial reaction centre’s primary electron donor.
Chlorophyll, pheophytin and quinone are parallel to the membrane.
And the same basic structure is found in the core of the photosystem I.
And you see here also primary electron donor and then you have one branch which is used for electron transfer.
And this also means that photosynthesis was invented only once during evolution,
but this happened more than three billion years ago already.
Now I change gears a little bit and I come to method development.
And method development is very, very important in research and what we did,
we tried to use fragments of monoclonal antibodies for membrane protein crystallisation.
The idea behind this that the membrane protein surface can be,
a surface which is polar, can be increased by the antibody fragments.
And it might be more easy to get crystals.
And this idea works out and you have heard Brian Kobilka
that he now uses also this trick in getting big crystals, in getting well-ordered crystals.
We used this primarily in the respiratory chain that is where we do substantially work on, respiratory chain.
You have here complex I.
You have here a fumarate reductase which is Complex II.
We have here Complex III, cytochrome bc1 complex.
We have complex IV which is cytochrome C oxidase and we have ATP synthase.
And the field is quite advanced.
You see that we now know the basic structures of all the components.
And actually these three structures were made by my group in Frankfurt
and these structures and these structures are made from Cambridge in the UK.
So we have a nice friendly competition between Cambridge and Frankfurt with that respect.
These are crystals of cytochrome C oxidase from a soil bacterium and basically that’s the part in the membrane.
So most of it is here in the membrane and that is the antibody fragment bound to a polar surface, enlarges the polar surface.
And what you also can see when you look at the crystal lattice
that actually the antibody fragment here glues together this cytochrome C oxidase.
There´s no direct contact between cytochrome C oxidase.
All the contacts are mediated by the antibody fragment here.
So this trick helps, it`s still tedious but you have a pretty good chance to be successful at the end.
So that’s method development.
Let me say I'm quite often asked: “How can you get Nobel Prize
when the governments are interested in getting Nobel Prizes for their countries?”
And I say to them that the most straightforward thing is method development
because all major methods for all major method development people have received a Nobel Prize.
So we have many examples here.
NMR, Electron Microscopy or DNA sequencing, everything got a Nobel Prize.
So inventing a novel important method I'm sure you will get a Nobel Prize.
Now I come to my favourite subject which is the cytochrome C oxidases.
They are part of the super-family of heme-copper terminal oxidases.
They perform reduction of molecular oxygen.
For that you need an oxygen molecule.
You need four protons.
They all come from the inside.
You need four electrons donated here from cytochrome C formed to water, four oxidised cytochrome Cs.
This happens in the cytochrome C oxidase.
And what is here in particular is that the electrons come all from the inside,
from the outside of the organelle or the bacterium and the protons come from the inside.
But in addition nature has invented a trick to pump 2-4 protons across the membrane.
So this of course leads to the generation of an electric voltage, so called membrane potential.
And also pH creatine, but the pH creatine is less of importance than the electric voltage.
So that’s the overall reaction that we’d like to understand in our research.
I will not go into details, too complicated.
Evolutionary tree of heme-copper oxidase is shown here.
We have three families and so we actually work on paracoccus,
a soil bacterium which is very close here to mammals, to bovine cytochrome C oxidase.
And it’s splitting occurred rather late.
I also will show you something about Type C, some kind of structure during the rest of my talk.
So these cytochrome C oxidases, terminal oxidases are quite diverse.
They are also pretty old in evolution and the question is:
Why are there enzymes reducing oxygen to water when there was no oxygen present three billion years ago?
And actually we don’t know what the original function of these terminal oxidases was.
It was clearly that they had originally some detoxification function
when oxygen became available due to the action of the water splitting photosynthesis.
So basically we have two copper atoms in A and B families and we have here a heme,
here we have copper, another heme group, and here is the site where oxygen is bound and reduced.
The protons come straight forward in this kind and the way as protons are pumped come via this case.
It’s not true for the other two families where we’ve only one channel which provides direct access to the active site.
But nevertheless this ends up now able to pump protons.
And that is really an enigma and we don’t understand that or how it works.
So this is basically our cytochrome C oxidase.
So here we have in the centre the active site where oxygen is bound and reduced.
We’ve electron transfer from cytochrome C bound to the oxygen here.
And we have proton transfer here and via that pathway to elective site and water is formed and protons are pumped in addition.
We also work on the C family, C family cytochrome C oxidases, the so called CBB3 oxidase.
And I have to say they are essential for many pathogenic bacteria, for instance helicobacter.
And if you delete the genes coding for the cytochrome C oxidase in helicobacter, helicobacter is no longer infectious.
So blocking this site of CBB3 oxidase would be a good way to kill the bacteria and cure stomach ulcers and gastritis.
So that’s another idea we work with.
So here only the central sub-unit I is similar to the A and B families
but in the rest then we have cytochrome molecules replacing the copper containing proteins.
And here we have a soluble electron donor getting cytochrome C
and the electron transfer then is down here towards the active site.
The electron transfer here is pretty fast
and they are much more efficient in oxygen reduction than the other type of cytochrome C oxidases.
A little bit of background how we get the stuff.
We isolate that from the native cells.
And you see here the various protocols, the various crystals which were obtained, which were disordered.
And I have to tell you that getting the purer stuff was not very good because the pure stuff doesn’t get well ordered crystals.
You would probably remove too many of the lipids and finding this out took one person, Sabina Buschman, ten years.
So still doing this kind of research sometimes you needs patience not only of the researcher but also of the funding agency.
Which you have to tell the funding agency that you really need... that they have to be generous in giving you time and money.
So with respect to the lipids I mentioned that we determined the lipid contents
and they’re all generally with about 50 lipid molecules per oxidase complex after an ion exchange chromatography.
That’s reduced to 12 lipids during the purification.
And further on by an isoelectric focusing reduced that to 6-9 lipids.
But the problem is this gives bad crystals whereas this preparation gives good crystals.
So the last purification step had to be removed in order to get the well diffracting crystals.
Now I change gears and I come to the sulphide quinone reductase.
And of course sulphide is toxic.
So you need in samples sulphide detoxification
because sulphide binds to the prophyrins of cytochromes and haemoglobin and it’s very, very toxic.
And also you have in higher animals, in mammals you have sulphide signalling.
And sulphide was proposed and actually is a gasotransmitter.
Very much it’s evolved from smooth muscle relaxation, very much like nitric oxide.
And you will hear more about that in the talk, which is going to be presented by Ferid Murad.
So we work on that enzyme and we got the structure in this case from a hyperthermophilic bacterium.
You see it’s a trimeric enzyme, it’s rather flat and it lies flat on the membrane, dives into the membrane.
And the membrane interaction you can see from the structure.
Down here we have some detergent molecules, some lipid molecules.
And we have tryptophan side chains which normally lead to the interaction with the membrane.
What we also have discovered quite astonishingly
that the enzyme binds the enzyme binds the flavin coenzyme in a very unprecedented way not seen before.
So here we have a disulphide bridge, two bridges connect and bind in covalently the flavin moiety to the protein.
And what we also found to our surprise was that you find a sulphur S8 ring in the protein.
The product of the reaction when you reduce the sulphide is sulphur
and actually it forms a ring and there’s one time when the sulphur binds to the cystine residue.
Was completely unexpected and not observed before.
We also could identify the quinone reduction site by adding quinones.
So quinones actually are the electron acceptor getting the electrons from the sulphide
and so that reduced quinone is the second product.
And so the overall mechanism of action can be seen here.
That is the enzyme lying flat on the membrane, diving into the membrane by about 12 angstrom.
And then you have access for H2S or HS- via nicely observed channels.
And from here...
You can here form the S8 ring.
You have channels for the excess for ubiquinone, the second substrate,
and the release of the product ubiquinonol which can be then used in the respiratory chain and be oxidised.
And the S8 ring can be or will be displaced through the membrane
inside to the bacteria and the bacterial world or into the mitochondrian mammals
because this enzyme is located on the outside of mitochondria in humans and of course also then in other mammals.
Of course we have to ask then the question: How is H2S... is hydrogen sulphide generated in mammals?
And there are two enzymes doing that.
One is cystathionine beta-synthase
and the other one is mercaptopyruvate sulphur transferase in combination with a cysteine amino transferase.
But the regulation of the production of hydrogen sulphide is unknown.
Then you ask what is the product in mammals because nobody has observed so far.
Sulphur globules in humans but actually it’s a thiosulphate.
So your body produces the inorganic compound thiosulphate.
And this happens by the sequential action of a sulphur dioxygenase and then the first product there is sulphite.
And then you have a sulphur transferase which adds a sulphur atom to the sulphide and you have thiosulphate.
And both these enzymes actually are located in the mitochondrian matrix.
But then further on how the thiosulphate is exported, how you get rid of thiosulphate is unknown,
what’s the further metabolism of that product.
So there are still some basic challenges in order to become known.
Let me say a few general words now about membrane protein structures.
And at present there are 484 membrane proteins where the structure is known.
That’s about 1,500 coordinate files.
And you see here in 1986 we had the first structures and it started off pretty slow.
And it was two structures only 5 years later.
But then people actually learned how to crystallise membrane proteins.
But I have to say it’s not primarily a question of improving the techniques.
Primarily it has been a question of selecting the more crystallisable... the better crystallisable membrane proteins.
Still when you work on your own membrane protein,
selected membrane protein it can take you ten or twenty years in order to get it.
But people nowadays they look for most stable variants and they sometimes get crystals within a few months.
This is why we have to progress.
But still progress with human membrane proteins is slow and there are for the moment about 40 human membrane proteins known.
Your body contains about 6,000 to 7,000 membrane proteins.
So there is still a lot to do before you actually can do at the end drug design
in order to cure diseases for the membrane proteins from the human body.
The methods for membrane protein structure determination are listed here.
Nuclear magnetic resonance can be used for some small proteins,
for more or less one or two transmembrane helices or some small beta-stranded membrane proteins.
Solution NMR 40 structures, solid state NMR 2 also made its way into membrane proteins.
There are two structures, then we have crystallography and it is x-ray crystallography
where most of the structures have been determined.
Electron crystallography is 7 structures.
But actually 5 of the 7 have also been determined by x-ray crystallography
and the quality of the structures determined by x-ray has been better than that by electron crystallography.
So x-ray crystallography has been the winner.
So where will be the future?
I still think it will be x-ray crystallography.
You may have heard that there are big machines now, free electron lasers.
There’s one operating at Stanford and another one is under construction in Hamburg.
There’s one going into operation soon in Japan.
And the idea is that you may be able to do diffraction with individual molecules at the end.
So you wouldn't need to crystallise that.
But this is still a dream.
And the idea behind it is that you can record a diffraction pattern of an individual molecule before it explodes.
So you have a very, very short x-ray pulse and the electrons of course interact with the x-rays.
They are blown away. But the diffraction is there.
But the molecule has been gone and you have a free plasma.
And so this is maybe the future, so we wouldn't get crystals.
But I fear that will be a dream.
Still the open question which I didn’t cover was that for mechanism.
I discovered that in the case of sulphide quinone reductase without going into the atomic details.
But diffraction methods provide static pictures.
In order to get the mechanisms of action you have to get structures of catalytic intermediates.
You have to analyse variants and you have to use various kinds of spectroscopy,
maybe photon transformation infrared spectroscopy, maybe EPR, maybe NMR.
But this depends on your protein.
And I'm satisfied when I find out the mechanism of a protein going really into the depths.
As I said there are still lots and lots to do
and I hope you will be interested to work on these interesting questions.
With that I want to end and thank you for your attention.