Session 3: G-Proteins

Transcript of Part 2: GTPase Reactions and Diseases

00:00:00.14	Hello. My name is Fred Wittinghofer.
00:00:02.18	I'm an emeritus group leader at the Max Planck Institute for Molecular Physiology in Dortmund, Germany.
00:00:11.08	And this is my second seminar
00:00:14.01	and in the first one I introduced you to the molecular switches called GTP binding proteins or G proteins.
00:00:21.15	And in this second part I will more concentrate on a particular aspect of our research which is
00:00:28.20	dealing with the mechanism of GTPase reactions and how they lead to a number of different diseases.
00:00:34.09	And I will obviously focus mostly on Ras-like proteins that I talked about in my first seminar.
00:00:42.23	So, again, just briefly, the mechanistic cycle for these proteins:
00:00:49.25	They come in two flavors these proteins; the GDP-bound and GTP-bound state.
00:00:55.13	They have nucleotide bound very tightly.
00:00:58.03	They need a guanine nucleotide exchange factor to release GDP for GTP
00:01:03.23	because in the cell, there is more GTP than GDP.
00:01:06.11	That's why the protein become loaded with GTP once GDP is off.
00:01:10.06	They have a downstream affect in the GTP bound form; interacting with some partner proteins.
00:01:15.10	And in order for the switch to be switched off again, you have the GTPase reaction
00:01:20.27	whereby the protein splits GTP into GDP and Pi.
00:01:24.29	And this reaction is very slow and is catalyzed by proteins called GTPase Activating Proteins
00:01:31.06	which will obviously be the major thing that we will be talking about.
00:01:36.24	So what we're talking about, really, is a really basic biochemical reaction;
00:01:43.23	namely, the hydrolysis of phosphoanhydrides and it's similar to the hydrolysis of phosphoesters.
00:01:53.08	For example, when you have phosphorylated protiens which are phosphorylated on the threonine, serine or tyrosine,
00:01:57.24	you have a similar nucleophillic attack on the phosphate by water.
00:02:05.04	And obviously, people have been talking about this reaction a lot because it is a very
00:02:12.15	slow reaction for the reason that the phosphates are highly negatively charged
00:02:19.16	and the approach of a nucleophile, for example a water which is partially negatively charged also,
00:02:25.06	is very, very disfavored. And that's why this reaction is normally very slow.
00:02:30.25	So even though the reaction delivers energy,
00:02:33.17	it is very slow because you have to overcome the very high activation energy
00:02:38.16	which depends on what I just told you about--the negative charges.
00:02:42.04	And the higher the activation energy, you might know from your biophysical courses,
00:02:46.13	that the higher the activation energy, the slower the reaction,
00:02:51.00	because the reaction rate is directly proportional to the activation energy.
00:02:54.18	So, nature, then, uses enzymes to lower the transition state energy
00:03:00.27	and thereby make the reaction faster.
00:03:03.15	And there's an interesting article that I bring to the attention of my students all the time
00:03:07.21	from Francis Westheimer who wrote an article many years ago:
00:03:12.01	Why Nature Chooses Phosphates.
00:03:13.25	Because chemists never use phosphate as a leaving group
00:03:17.13	but in biology it is a very frequent leaving group
00:03:21.16	So, because of just this purpose here, because of just what I showed here
00:03:26.17	the phosphoanhydride bond or the phosphoester bond is kinetically stable
00:03:32.06	in other words you can have your ATP or GTP in water and it stays forever or hydrolyzes very slowly.
00:03:39.22	But thermodynamically it's principally unstable.
00:03:43.00	If you use an enzyme, it lowers the activation energy--you can make this reaction very fast.
00:03:47.14	So that's an interesting article that I would recommend you to read.
00:03:50.27	For example, that's why DNA is stable and that's why ATP is such a wonderful source of energy
00:03:57.12	because it, in principle, delivers energy but is stable in aqueous solution.
00:04:03.28	So let's start then with first of all Ras and its GAP-mediated GTPase reaction because
00:04:10.25	that is really the paradigm for many of the things that have been developed afterwards.
00:04:15.24	So this is the molecular Ras. Its a ribbon representation of the structure
00:04:21.29	of the G domain of Ras.
00:04:24.16	You see its heart shaped because...
00:04:27.17	obviously we like it very much and we solved the structure in Hiedelberg.
00:04:32.00	The city of Hiedelberg's advertisement, Ich hab mein Herz in Hiedelberg verloren
00:04:36.04	which means, I lost my heart in Hiedelberg.
00:04:37.27	But the most important reason it's heart shaped is because
00:04:41.13	people call it the beating heart of signal transduction.
00:04:44.06	And so its one of the most important molecules that regulates
00:04:48.14	important signal transduction processes like growth, differentiation and sometimes even apoptosis.
00:04:54.17	And you probably know a little bit about the signal transduction process because
00:04:59.07	every text book has this version, one of the paradigms of signal transduction chain
00:05:04.01	whereby, for example, a growth factor binds to the cell membrane
00:05:08.27	and binds to its growth factor receptor RTK (receptor tyrosine kinase)
00:05:14.22	whereby this becomes phosphorylated. It recruits the exchange factor SoS
00:05:19.04	which then activates Ras to the GDP bound form.
00:05:22.16	And then, now, Ras interacts with the downstream component which is Raf kinase
00:05:26.15	which is the starting kinase for what is called the MAP kinase module.
00:05:30.24	There, one kinase activates the next downstream kinase which is called MAP kinase kinase kinase.
00:05:36.29	MAP kinase kinase kinase activates MAP kinase kinase and that activate MAP kinase
00:05:40.28	which then goes into the nucleus and activates transcription.
00:05:45.03	So this is a very simplified version of what Ras is actually doing.
00:05:48.16	and this is the first one to be discovered (the first signal transduction).
00:05:54.15	But, now it becomes more and more complicated.
00:05:57.26	This is still a very simplified version but it shows you already
00:06:00.27	the major thing about signal transduction via Ras in that many upstream components come and activate Ras.
00:06:09.18	It's either tyrosine kinase receptors or G-protein coupled receptors
00:06:13.09	or T-cell receptors. All of them can activate Ras.
00:06:16.08	And then Ras can activate, downstream, many components, not just Raf kinase
00:06:20.18	but also a molecule called Ral GEF, PI(3) kinase, PLC epsilon and others.
00:06:26.22	And they mediate a number of signal transduction reactions
00:06:32.12	which somehow are integrated somewhere, let's say in nucleus,
00:06:38.00	by a transcription factor and initiate, when the threshold is right, when the number of reaction is right,
00:06:46.15	it initiates a cellular response which can be proliferation or differentiation or whatever.
00:06:52.03	And I will not deal with any aspect of this because I want to concentrate on the switch-off reaction.
00:06:57.28	So the switch-off reaction is again the scheme you have seen, now, many times
00:07:03.25	but what happens in Ras is also an oncogene;
00:07:06.26	an oncogene that has two types of mutations, either the glycine 12 mutated to any other amino acids
00:07:13.10	or glutamine 61 mutated to any other amino acid.
00:07:16.21	And what this does biochemically is, it blocks the GAP-mediated GTPase reaction.
00:07:23.21	So you can imagine what happens, you have blocked the return to the inactive state
00:07:29.13	and that's why you now accumulate Ras in the GTP bound form.
00:07:33.09	You don't need any of the upstream signaling anymore because Ras is already in the GTP bound state.
00:07:39.03	And now it has an effect that is not regulated anymore and that's why it leads to cancer.
00:07:44.14	So these simple mutations...and that's why, obviously, as structural biochemists
00:07:49.23	it is a very interesting project to ask the question:
00:07:52.00	How can it be that a single point mutation has such dramatic consequences?
00:07:58.19	And its also...obviously since Ras is the most frequent oncogene.
00:08:02.28	About 25% of the people that come to the clinic diagnosed with a tumor,
00:08:09.07	they have a Ras mutation, one of the ones I showed you. At least these are the most frequent ones.
00:08:15.00	So obviously every drug company also are working on trying to inhibit the Ras pathway
00:08:20.29	and Ras signaling as a way of treating Ras-mediated cancers.
00:08:25.28	And there are many approaches that one can think of.
00:08:30.17	I just indicated a few here. For example, you could think of Ras...is farnesylated
00:08:36.25	at the C-terminal cysteine and there's an enzyme called farnesyl transferase that mediates that reaction.
00:08:44.18	There are farnesyl transferase inhibitors that are just in the clinic being tested for their efficacy.
00:08:50.00	But you could also think of maybe inhibiting the interaction with downstream effectors.
00:08:54.15	This is, for example, a structure we determined: the complex between Ras and Raf kinase (or part of Raf kinase).
00:09:00.10	Or you can even think of...and that's what we're working on still...
00:09:04.21	Our dream approach is...the basic feature of oncogenic Ras is that is cannot hydrolyze GTP.
00:09:12.09	Can we think about making small molecules that would induce GTP hydrolysis on oncogenic Ras?
00:09:18.21	This is probably a dream project and we're still working on it to make it feasible.
00:09:25.05	But I will show you, in the course of my seminar presentation, that the reason we still think its possible
00:09:32.23	is that the chemistry of it should not be so difficult.
00:09:36.14	And I hope I can convince you and that you will go with me on that point in the end.
00:09:40.26	So, we are talking about, here, a nucleophilic attack of water on the gamma phosphate of GTP
00:09:49.03	mediated by RasGAP and it produces Ras-GDP and Pi. A simple biochemical reaction
00:09:56.09	but if it doesn't work it leads to very drastic consequences, namely cancer.
00:10:00.21	So first of all (And you have seen that picture also a number of times.
00:10:05.24	This is my introductory slide, always)
00:10:08.09	is that the two amino acids that are most frequently mutated
00:10:13.16	(either of them) in oncogenic versions of Ras, they are very close to the active site.
00:10:18.07	So you see the nucleotide here: that is the gamma phosphate
00:10:22.07	And you see this is glutamine 61 and this is glycine 12 are very close to the active site
00:10:28.10	obviously, as you would probably predict or thought of before,
00:10:32.06	is that they are somehow involved in the GTPase reaction.
00:10:35.22	Now, we'll obviously show you what they actually do and why the mutation leads to a block in GTP hydrolysis.
00:10:43.08	And you also can see here from a surface representation of the Ras active site
00:10:49.12	that if this GTP...so it sits...its bound to the surface
00:10:53.26	and you see the gamma phosphate is still approachable from the outside
00:10:57.03	and that will be important in the context of what I will be talking about
00:11:01.12	and you see also, I talked, last time about magnesium being important.
00:11:05.20	If there is no magnesium around you also have no GTP hydrolysis reaction.
00:11:09.24	So, as always in a mammalian genome, there are not just one RasGAP, but rather, 12 or 13.
00:11:19.08	And I indicated here a few of these.
00:11:24.06	And you see they all contain one particular domain of about 330 residues.
00:11:28.09	This is the RasGAP domain which by itself is able to initiate the fast GTP hydrolysis reaction.
00:11:36.17	And you see that all these proteins are composed of different domains.
00:11:43.03	And some are similar, some are vastly different.
00:11:45.20	And they obviously are regulating Ras in different contexts of the cell
00:11:50.04	due to their different domains that they have in addition.
00:11:53.15	And I will be talking about one, the first RasGAP to be discovered by Frank McCormick which is P120GAP.
00:12:02.06	And I will be talking later on about NF1
00:12:05.05	which is another important element for tumor formation because it is a tumor suppressor gene.
00:12:12.04	So first of all, and again to remind you, the intrinsic GTP hydrolysis is very slow.
00:12:17.23	But, if you add the GTPase activating protein, it is fast.
00:12:21.08	So what I have been doing here is I take radioactive GTP (gamma labelled, for example)
00:12:25.27	and then I measure the production of Pi over time.
00:12:29.26	You see, down there, this reaction (this is at room temperature) is almost negligible.
00:12:35.14	There's almost no hydrolysis of normal Ras at room temperature without GAP.
00:12:40.12	And if you now add a particular amount of RasGAP you see that there is a very fast reaction.
00:12:44.23	Much much faster.
00:12:46.04	And under limiting conditions it's actually about 10^5-fold stimulation of that reaction.
00:12:53.10	So that is a way we want to look at it.
00:12:57.05	We want to analyze how GAP mediates this fast GTP hydrolysis reaction.
00:13:03.14	So, initially, obviously, you ask yourself: What could be the mechanism of hydrolysis
00:13:10.11	and what is the step that is catalyzed by GAP?
00:13:14.02	You can think of Ras-GTP being, in principle, a fast GTP hydrolysis enzyme
00:13:20.14	but it needs to come into a GTPase competent state by an isomerization reaction.
00:13:26.15	And that is very slow and is catalyzed by GAP.
00:13:28.29	Or you could think that the actual cleavage reaction, going from GTP to GDP Pi,
00:13:35.02	is very slow and is catalyzed by GAP.
00:13:38.01	Or you could even think that all of that is still very fast
00:13:41.09	but the release of Pi to product is very slow and that is catalyzed by GAP.
00:13:47.08	For example, if you remember having heard about the hydrolysis of ATP on myosin,
00:13:53.26	myosin is, for example, a very fast GTPase but the Pi release is very slow
00:13:59.00	and needs to be catalyzed by actin.
00:14:01.04	So, in other words, what is the actual step that is catalyzed by GAP in this particular case?
00:14:07.10	And I should say that it is the cleavage reaction itself
00:14:11.12	which is the major point of attack by GTPase activating protein
00:14:16.21	and that only in the presence of GAP do the other reactions
00:14:20.18	become at least partially rate limiting, at least the Pi release.
00:14:24.10	And I'll show you that later on, as well.
00:14:27.29	And although I've shown you that before, I will repeat that again.
00:14:32.12	So what we use a lot in our studies where we do biochemistry with fluorescent nucleotide,
00:14:38.20	we use a mant- or mGDP or mGTP analog
00:14:42.29	where you have, on the ribose, a fluorescent reporter group.
00:14:46.24	So this would be either deoxy ribose or ribose and on the ribose you have bound by
00:14:52.17	these ester bonds, the mant group which is very sensitive to the environment that it sits in.
00:14:59.19	And that's why it always give very beautiful structural changes as I will show you in a minute.
00:15:07.10	We used stop flow for measuring fast reactions because, remember, the GTPase reaction,
00:15:13.29	which is slow in the absence of GAP becomes very fast
00:15:17.15	and so in order to analyze it in detail, we need to use stop-flow kinetics.
00:15:21.16	So what you'll, for example, do: you have Ras labeled with
00:15:25.10	mant-GTP (so it's the fluorescent version of GTP)
00:15:28.16	and you have GAP and you shot them together into a fluorescent-detection cuvette
00:15:34.02	and you have the stop flow up here in order to
00:15:37.17	make sure that you only put a certain amount of liquid from these two syringes into your reaction chamber.
00:15:44.07	So when we do that, when we shot these two things together, we see that there is
00:15:50.09	a fluorescent increase if you use Ras-mantGTP and a decrease.
00:15:54.09	The increase, again, is very fast.
00:15:56.25	It means the two proteins make a complex.
00:15:58.27	And then over time, the complex dissociates because after hydrolysis neurofibromine does not bind to Ras anymore.
00:16:07.18	And all of this is over, as you can see, after one second.
00:16:10.09	So, very fast phospho-transfer reaction in the presence of saturating amounts of GAP.
00:16:15.07	And as a control, we use Ras bound to an analog, GppNHp,
00:16:21.25	where you that between the beta and the gamma phosphate you have an NH group
00:16:25.16	which cannot be hydrolyzed anymore and now you have, also, a very fast increase
00:16:30.17	(which is complex formation) but no dissociation because that cannot be hydrolyzed.
00:16:35.05	So that is also the proof that the dissociation is due to GTP hydrolysis.
00:16:40.21	So one may ask, "In this reaction here, what on GAP, which residues on GAP,
00:16:50.01	are involved in mediating this fast GTP hydrolysis reaction, that 10^5 fold stimulation of the reaction?"
00:16:58.17	And obviously, you were thinking of which residues, which amino acids could do the job.
00:17:03.16	Or is it more than one amino acid?
00:17:05.16	And as a good candidate, we were thinking of an arginine
00:17:08.16	because if you look at another G-protein, G-alpha protein (of the heterotrimeric G proteins)
00:17:15.02	it is known that that consists of two domains;
00:17:18.25	so this blue domain is the G-domain and the red stuff here is a helical extra domain.
00:17:24.21	But in that helical domain you have an arginine which sits right, smack in the active site
00:17:29.24	and it has been shown that that arginine is important for the reaction.
00:17:33.29	And that has been shown, in a very old experiment
00:17:37.20	because there is a bacterial pathogen called Vibrio cholera which induces cholera
00:17:45.29	and what is does is it actually modifies this arginine on this G-alpha protein.
00:17:51.16	You see, you have NAD and the cholera toxin transfers ADP-ribose onto
00:17:58.03	an arginine of G-alpha which is shown here. This is actually from the textbook here.
00:18:02.26	So this a very old reaction. It had been analyzed many, many years ago.
00:18:07.10	And it shows, when you do this reaction, when you block
00:18:10.16	the GTPase reaction on G-alpha protein, there is no GTP hydrolysis anymore
00:18:16.10	and the protein is now producing cyclic AMP,
00:18:19.14	it opens up channels (cyclicAMP-gated ion channels) and then you get all these symptoms of cholera.
00:18:26.19	So again the question is: would arginine be a good candidate?
00:18:29.29	So we looked for conserved arginine in all the RasGAPs that I've shown you
00:18:33.24	and indeed, we found several, most of them didn't make any difference.
00:18:38.06	But there was only one arginine that when mutated shows the following pattern in the reaction.
00:18:43.19	So you, again, have in the green version (this is the wild-type version);
00:18:47.23	fast increase due to complex formation, fast decrease due to dissociation after GTP hydrolysis.
00:18:53.16	And with these mutants here, arginine mutated to either lysine or alanine
00:18:58.11	or anything you'd like to mutate it to,
00:19:00.03	there is again an increase in the reaction but then it stays up there, no hydrolysis whatsoever.
00:19:06.13	Or, at least under those circumstances, up to a second, there is no hydrolysis.
00:19:10.16	Which tells you already, obviously, that this arginine must be essential for the reaction.
00:19:18.02	So far so good. The biochemistry was clear
00:19:21.09	but obviously you don't really know what arginine is doing in the context.
00:19:24.05	You think you have an idea; it may be going into the active site
00:19:28.02	but, again, we have to wait for the structure to tell us really what it does.
00:19:33.21	First of all, though, let me introduce you to another important concept for analyzing phospho-transfer reaction.
00:19:40.12	And that is using aluminum fluoride complexes.
00:19:43.02	You may wonder why such an inorganic molecule would be important
00:19:47.14	but it turns out that if you look at the nature of the transition state,
00:19:51.20	so this would be GTP being attacked by water.
00:19:55.18	You have a transition state where the phosphate now makes a triginal, flat thing
00:20:00.26	where the nucleophillic oxygen and the leaving group oxygen on the other side
00:20:08.22	are the axial ligands of this penta-coordinate phosphate
00:20:12.11	and this transition state can either be very tight (which is then called associative)
00:20:18.16	or very loose depending on the distance here between phosphate and the two axial ligands.
00:20:25.26	And then you end up with, again, tetragonal phosphate (this Pi free phosphate).
00:20:32.08	And it turns out that aluminum fluoride
00:20:36.05	(which has distance between aluminum and fluoride very similar to
00:20:39.23	between phosphate and oxygen and they are also highly electron negative)
00:20:44.26	that this is a very good mimic of the transition state of the phospho-transfer reaction.
00:20:51.02	The only problem was that while, for example kinesin or myosin or
00:20:56.28	many other phospho-transfer enzymes use aluminum fluoride as a mimic of the
00:21:02.07	gamma-phosphate in the transition state,
00:21:04.17	Ras or Rho and all of these Ras-like proteins, never showed that.
00:21:08.25	Here you see that experiment.
00:21:11.19	You take Ras-mantGDP (it has  a certain fluorescent emission spectrum with a maximum at around 440)
00:21:19.03	and now you add the GAP and with one there's no change whatsoever to the spectrum.
00:21:24.12	So nothing happens.
00:21:25.11	But if you now add aluminum fluoride, you see that now you get a blue shift first of all
00:21:31.00	and then an increase in the absorption.
00:21:37.11	That means there is a trimeric complex between Ras, NF1 and aluminum fluoride
00:21:42.07	where the aluminum fluoride sits in the gamma phosphate binding site.
00:21:47.10	And that was very instrumental...
00:21:48.25	and by the way, if you now do this in oncogenic mutants
00:21:52.02	which we know does not hydrolyze GTP and do the same experiment,
00:21:55.15	you see, in the presence of aluminum fluoride and Ras, there's no fluorescent change.
00:22:00.06	And now you add NF1 and there's no increase in fluorescence
00:22:04.12	because you have a mutation (Q61L) that does not hydrolyze.
00:22:08.13	You can also take a mutation of NF1, for example with the arginine mutation,
00:22:13.22	and again there would be no change whatsoever.
00:22:16.05	So in other words, what these experiments again tell us is that Ras in an incomplete phospho-transfer enzyme.
00:22:22.22	It needs the presence of a GAP in order to look like a phospho-transfer enzyme
00:22:28.05	and if there's any mutation that blocks the GTPase reaction, either on Ras or on NF1,
00:22:33.22	we also get no aluminum fluoride complex.
00:22:37.09	So obviously, all of this is nice in terms of doing biochemistry
00:22:42.00	but what really mediates the GTPase reaction, the fast one,
00:22:47.22	we think can only be verified by looking at the structure of the Ras and RasGAP complex.
00:22:55.29	This is shown here. So that was the paradigm for analyzing this type of reaction.
00:23:02.06	So you see, for example, in red you see Ras and down in green is the RasGAP domain (P120 GAP).
00:23:09.13	And what you see then, here...if you look very carefully
00:23:12.04	you see that there is a residue from GAP going into the active site.
00:23:17.14	You see that when it comes back...you see that right there.
00:23:21.01	There is an arginine residue pointing into the active site of Ras
00:23:26.04	and what is does is shown in the next slide.
00:23:29.06	First of all, in this slide it shows the aluminum fluoride really is the flat triangle
00:23:34.00	that sits between the leaving group and the nucleophillic water.
00:23:38.07	So there is a mimic of the transition state.
00:23:39.28	And you see then, also, what are the residues that mediate fast GTP hydrolysis.
00:23:45.17	So this would be the phosphate,
00:23:48.14	so we think now that if we take away aluminum fluoride and
00:23:52.06	think of what the real transition state would look like
00:23:55.06	you have the nucleophillic water and you have the transition state
00:23:58.29	where they are bound to the gamma phosphate
00:24:00.25	and the glutamine is fixating the water relative to the phosphate
00:24:05.07	by an acceptor and a hydrogen bond donor interaction.
00:24:09.17	And the arginine (what we call arginine finger) is first of all stabilizing
00:24:13.29	the position of that glutamine and it also delivers this positive charge
00:24:18.03	of the amino group to neutralize charges in the gamma phosphate.
00:24:22.28	So these are the two residues that are really crucial for the reaction;
00:24:27.08	glutamine 61 from Ras and the arginine finger from RasGAP.
00:24:32.05	And this already explains, for example, why mutations of glutamine 61 in oncogenic Ras
00:24:37.19	mess up the hydrolysis reaction because you can imagine if you have, for example,
00:24:42.09	a leucine or whatever here, if cannot do this kind of interactions here.
00:24:47.00	So any mutation of glutamine 61 is oncogenic because it is a direct catalytic residue.
00:24:53.16	So the structure also explains why mutations of glycine 12 are oncogenic.
00:24:58.22	And that also can be easily seen here on this slide.
00:25:02.04	You have glycine 12 which when mutated to any residue makes it an oncogene.
00:25:06.25	And then you see aluminum fluoride being this flat triangle.
00:25:11.08	You see glutamine 61 being there and you see the arginine finger down here.
00:25:16.09	And they're all very very close together indicated by these blue dashed lines
00:25:20.04	which are indicating that these are almost VanDerWaals distance.
00:25:24.04	And now if you mutate, for example, glycine to the smallest possible amino acid,
00:25:28.11	it would be alanine, you see that it would immediately mess up all these residues in the active site.
00:25:34.01	So for steric reasons, there can be no other residue
00:25:36.24	in the active site in the transition state except for glycine.
00:25:43.08	So the message from all of this was, obviously, that there's an arginine
00:25:49.00	that we call the arginine finger that pulls the trigger on the GTPase reaction.
00:25:54.00	Without it, it is very slow and with it, it becomes 10^5 fold faster.
00:26:01.01	We used also...so coming back to the question: what is now rate limiting in the whole process?
00:26:07.00	So the main regulating step of intrinsic GTP hydrolysis is the very slow chemical step, the slow hydrolysis.
00:26:14.29	But using a different technique we now find out that
00:26:17.25	there is another step that become partially rate limiting and I will show you that in a minute.
00:26:23.29	So we use time-resolved Fourier transform infrared spectroscopy.
00:26:28.10	And with that we can do almost atomic resolution.
00:26:33.11	We can look at atoms in the active site on a millisecond timescale.
00:26:37.27	So if you see an infrared spectrum of a protein, which has amide I and amide II bands,
00:26:47.20	which is not very structured information because its a mixture of information on
00:26:54.26	alpha helices, beta sheets and so on and not a great deal of detail can be taken from such a picture.
00:27:02.26	But what we do is we observe, for a reaction...let's say a protein goes from A to B
00:27:09.05	we observe the different spectrum which at least on this absorption scale
00:27:16.19	does not show much of a difference but if you look in more detail...
00:27:20.15	so you see the absorption there is 0.0 and the absorption down here is 0.02,
00:27:25.14	so we see very small but very reproducible changes
00:27:29.21	in the course of the reaction when A goes to B.
00:27:33.02	And we see negative peaks that mean A goes away and we see positive peaks if B comes up.
00:27:40.22	So we can observe things that are lost and things that come up
00:27:43.29	in the course of the reaction and we can follow these by FTIR.
00:27:49.25	And so if you do...so first of all we need to trigger the reaction at a particular time point
00:27:56.25	in order to observe second timescale structural changes.
00:28:03.04	And in order to do that we use, again, a different analog
00:28:07.00	and the different analog is caged GTP which was developed by Roger Goody
00:28:10.22	who is a colleague of mine in the Institute.
00:28:14.05	And this caged GTP is blocked on the gamma phosphate by what is called the cage group.
00:28:20.22	So it doesn't allow hydrolysis but now, with a flash of light, you can cleave that cage group off
00:28:26.21	and now you have Ras-GTP which can then hydrolyze to GDP and Pi.
00:28:31.19	If you do that with let's say Ras without GAP,
00:28:39.11	what you see is that you have a...for example, you see absorbance...
00:28:44.22	so any absorbance that you might know from infrared, shows an atomic vibration in the bonds.
00:28:51.09	For example, you see vibrations for the
00:28:53.15	alpha, the beta, the gamma, which decrease toward the end and become zero after 2 hours, 5 minutes.
00:29:00.29	So the difference spectrum (subtract the end spectrum from the starting spectrum)
00:29:06.16	and then you see only the changes that are occurring during the reaction.
00:29:11.23	And that is quite normal. There is a single exponential decay when Ras hydrolyzes GTP.
00:29:18.21	No big deal.
00:29:20.15	But what is interesting is that when you analyze the GAP mediated reaction
00:29:24.26	because now you don't see a single exponential decay, but you suddenly see intermediates appear.
00:29:30.14	You see peaks that come up and go down and the most important one is
00:29:35.04	indicated by this number 1113. This is the frequency for that particular change.
00:29:39.28	So you obviously have to analyze what is each band doing, what does it belongs to.
00:29:47.06	So what we're doing in order...we have developed these techniques
00:29:52.17	or our colleagues at the University of Bochum with whom we collaborate
00:29:56.03	they have developed techniques to find out what is each bandwidth,
00:30:01.01	what is each frequency...absorbance change...what is it due to.
00:30:03.28	For example, you find for GTP that there is absorbance change coming up with
00:30:10.13	a rate constant k2. So k1 is the photoisomerization, k2 is one reaction and k3 is the next one.
00:30:17.18	And if you do that, you get an increase and a decrease with k3.
00:30:21.12	And then there is an intermediate which is coming up at 1113.
00:30:26.12	It comes up with k2 and decays with k3.
00:30:30.01	There is free Pi coming with k3. From all of that we can obviously conclude
00:30:35.18	that there is a Pi intermediate with this absorption frequency here, 1113 cm^-1
00:30:44.02	which appears with the rate constant k2 and decays with a rate constant k3.
00:30:50.03	So in other words, the release of Pi is now  becoming visible.
00:30:54.25	So we see hydrolysis when this Pi peak comes up.
00:30:58.21	And we see decay when it goes away.
00:31:01.01	And you see this here, for example, in real-time.
00:31:03.25	So the absorption change with the rate constant k2...you see the scheme here:
00:31:09.05	Ras when its in the on state goes to the Pi state and you see there is a protein bound Pi
00:31:17.13	that comes up and the Pi band goes down in the course of the reaction.
00:31:21.20	And this is repeated a number of times.
00:31:23.07	And you also so that there is an arginine finger that is coming in and out of the reaction chamber.
00:31:30.00	So we can follow not just protein bands,
00:31:32.16	we can follow the phosphate bands at atomic resolution on a milli second timescale.
00:31:40.26	And that tells us now, all together, that is the message from all of this,
00:31:46.01	that while you have a high activation energy for the intrinsic hydrolysis reaction of 92kJ/mol
00:31:54.01	you now seperate the reaction in two partial reactions,
00:31:57.16	which have a lower activation energy and that's why is makes it so much faster.
00:32:01.08	So you have the first activation energy for the cleavage reaction. which leads to Ras-GDP-Pi.
00:32:08.03	And you have the second step where you have release of Pi and now you have the product.
00:32:12.14	So that is, again, a general theme of enzymology;
00:32:15.12	that an enzyme catalyzed reaction lowers the activation energy
00:32:20.00	not just by lowering one reaction but also by subdividing it into partial steps
00:32:24.28	each of which has a different activation energy.
00:32:29.22	And here you see that this activation energy is 59 kcal/mol and 66 which means
00:32:36.04	that this is a bit higher and that's why this is the partially rate limiting step of the overall reaction.
00:32:45.12	So let me now give you...so that was Ras and how it leads to tumor formation
00:32:50.14	and we analyzed in detail how this function even on a biophysical level
00:32:55.02	but now let me come back to why certain GTPase reactions, when the don't work,
00:33:03.00	how they lead to different types of diseases.
00:33:05.20	Neurofibromatosis is one of them.
00:33:07.27	I already showed you that neurofibromine, the gene product of the gene is a Ras GAP.
00:33:15.09	And there is a disease called Type I Neurofibromatosis. Its what people have cafe au lait spots on the skin,
00:33:22.21	sometimes small tumors on the skin which can sometimes be rather large and very disfiguring
00:33:28.16	which are caused by mutation of deletion of neurofibromatosis gene.
00:33:35.21	And for example, when we worked on the mechanism of GTP hydrolysis, by GAP and NF1,
00:33:43.00	a colleague from the Charite Clinic in Berlin came to us and told us that he had
00:33:48.03	a female patient which dies at teh age of 35 and she has three sons indicated up there
00:33:55.20	which also have the disease. And he analyzed the blood of the patient and then the tumor itself.
00:34:04.22	He found out that there is a mutation in the sequence of neurofibrobromine
00:34:11.29	where the arginine is mutated to proline. You see that down there; arginine is mutated to proline.
00:34:17.13	And obviously I wouldn't tell you that, if it wasn't the catalytic arginine.
00:34:21.19	So it turns out they have a mutation in the catalytic arginine
00:34:25.23	and when you now do the GTPase reaction that I've shown you before,
00:34:30.09	you see that you have, with normal NF1 you have the blue curve here,
00:34:37.17	it means an increase in fluorescence and a decrease with hydrolysis
00:34:43.03	and now if you take this mutation, R to P, you have increase which means complex formation
00:34:48.03	but no hydrolysis and there is another patient that we've analyzed in the meantime
00:34:53.16	again arginine to anything else, Q in this particular case,
00:34:58.09	leads to a block of its ability to hydrolyze GTP on Ras.
00:35:02.17	So mutation of the essential arginine leads to the disease neurofibromatosis.
00:35:08.16	Also, I should point out that there are many other mutations in neurofibromine
00:35:13.22	which also cause the disease which phenotypically very different in many different patients.
00:35:20.26	Let me introduce you now to a different system that is
00:35:24.06	interesting both from a biochemical standpoint but also from a standpoint of a different disease
00:35:29.11	that I will come to in due course.
00:35:33.03	So what I will be talking about is a molecule called Rap which, obviously, has a cognate RasGAP.
00:35:40.16	And Rap is close homolog of Ras and the name derives from
00:35:44.05	the fact that it is highly homologous to Ras because Rap stands for Ras Proximate.
00:35:49.07	Even though it was considered to be a close homolog of Ras, it does something completely different.
00:35:55.14	It obviously has the same cycle between GDP and GTP. So it works as a molecular switch.
00:36:02.00	But it does not have to do with proliferation, as Ras, or differentiation, but rather
00:36:06.10	it is involved in integrin activation, or platelet activation or other things.
00:36:11.19	So the biology of Rap is completely different.
00:36:14.17	Why the biochemistry is so interesting is that Rap is the only homolog
00:36:22.12	or the only member of the Ras super family that doesn't have a glutamine in the position
00:36:27.18	where glutamine 61 of Ras is involved in GTP hydrolysis.
00:36:33.01	So, it misses the residue that we thought was absolutely crucial GTP hydrolysis and here, its not there.
00:36:39.18	And, obviously, the question is why that is so and how does RapGAP
00:36:42.25	then work on this system and how does it stimulate the reaction.
00:36:48.29	So first of all, let me introduce you to the RapGAPs.
00:36:52.24	There are indicated here five of them but there are more in the human genome
00:36:57.23	but these five RapGAPs all contain a domain that is highly homologous
00:37:04.02	which is indicated here by the light blue and the dark blue staining
00:37:08.09	where the light blue stuff is somewhat different to the dark blue.
00:37:12.05	And I will explain that when we look at the structure.
00:37:13.21	So again, the domain organization of all these five RapGAPs is somewhat different.
00:37:17.25	That means they're probably doing their job in a different biological context.
00:37:23.04	The reason its also interesting is that the dark blue homology region is
00:37:28.10	also conserved in a protein called Tuberin
00:37:30.17	which stands for a disease that I will be talking about in the end.
00:37:34.03	So that's why it was also interesting to look at that reaction.
00:37:37.06	And the third reason its interesting to look at that reaction is indicated in the next slide.
00:37:42.19	But before doing that, let me show you first of all that we do, again, a stopped-flow fluorescence assay.
00:37:48.23	We've developed a system where we can look at this reaction in a biochemical way
00:37:53.12	and analyze mutations and the speed of reaction and so on.
00:37:58.13	So here again, we have Rap-GTP interacting with RapGAP
00:38:03.20	You get a large, quick fluorescent increase which is due to complex formation
00:38:07.28	and a decrease due to GTP hydrolysis and dissociation of the product.
00:38:12.25	And it is all over, again, after one second.
00:38:16.03	whereas without RapGAP, the whole reaction would take hours.
00:38:19.25	So, in other words, we again have 10^5 stimulation of the reaction.
00:38:23.15	But the reason this is also an interesting system biochemically and mechanistically,
00:38:29.09	is that there are a number of conserved arginines in RapGAPs and obviously
00:38:35.00	we thought that one of them would be involved in providing an arginine finger to the system.
00:38:39.06	In fact, we mutated all of them to alanine
00:38:42.06	and none of them has any dramatic effect on activity as one can see here.
00:38:48.02	So the worst reaction is still close to .5/second.
00:38:52.02	So in other words, there is not a dramatic effect when you mutate an of the arginines
00:38:55.29	which makes it unlikely that there is an arginine finger involved in the reaction.
00:39:00.23	So the two residues, intrinsic glutamine and the arginine finger in trans
00:39:09.04	which is Ras and Rho and others make the important catalysis are not here in this system.
00:39:17.05	That means the whole chemistry must be totally different.
00:39:21.15	So we looked at the FTIR of the system again and
00:39:26.07	it shows basically the same features even thought their structures are somewhat different.
00:39:31.16	The basic features are that there is a Pi intermediate whose decrease is rate limiting
00:39:37.23	for the reaction and although its chemically somewhat different,
00:39:41.27	as the different absorption spectra show, it is, indeed, kinetically a most important intermediate.
00:39:50.11	But what was interesting, and we found in this particular case, in the RapGAP case,
00:39:56.10	and it was also found in other systems in the meantime,
00:39:58.27	is that the GTPase reaction is reversible,
00:40:01.29	which sounds sort of crazy because it's a downhill reaction.
00:40:05.06	If you take GDP and Pi and the whole system you would never ever create GTP.
00:40:09.22	But what happens is that if you analyze the reaction by
00:40:13.05	using, instead of normal water, O18 water, which is indicated by this black dot,
00:40:17.23	you would expect that in the reaction you get hydrolysis and then you get GDP and Pi
00:40:23.27	which has one phosphate labelled as O18 oxygen-rather, one oxygen labeled as O18 oxygen.
00:40:30.12	But instead of getting a Pi with one O18 oxygen, you get Pi with two O18 oxygens,
00:40:36.24	with three O18 oxygens, and with four O18 oxygens as analyzed here by a mass spec.
00:40:42.11	You see where you get all these four products, analyzed by mass spectroscopy.
00:40:49.27	So, how does this happen?
00:40:51.09	So it happens because on the protein you have this long-lived intermediate
00:40:56.00	with GDP and Pi, sitting in the active site before going into product that can also
00:41:03.10	do a back reaction to make GTP with one oxygen now on the gamma phosphate.
00:41:08.15	Now, if it reacts again with O18 water, you get incorporation of a second O18 into the product.
00:41:15.27	And if it happens again, a third one and a fourth one.
00:41:18.16	So although the overall reaction is downhill,
00:41:22.12	on the enzyme, you're getting a backwards reaction.
00:41:24.15	And you can never get it once Pi is released
00:41:28.21	because then the activation energy for the reverse reaction is too high.
00:41:33.18	So we solved the structure of RapGAP also.
00:41:37.06	This is a two domain structure where one domain, which you see now to the left,
00:41:44.07	is the catalytic domain--the dark blue region in the homology diagram that I showed you
00:41:49.01	and the light blue stuff is the dimerization domain which is not important for catalysis
00:41:53.08	but is there to dimerize the protein
00:41:56.18	for whatever reason that we really do not know.
00:42:00.13	So in analyzing the catalytic domain, obviously, you ask yourself:
00:42:03.10	What are the conserved residues and which of those play an important role in catalysis?
00:42:08.04	The purple helix that I've indicated here is the most highly conserved region.
00:42:12.00	So it's likely that residues on this helix are somehow involved in catalysis.
00:42:18.05	And indeed, you can mutate many of these and you see certain effects.
00:42:21.12	But the most dramatic effect happens when you mutate
00:42:25.01	N290, so an asparagine, sitting on this catalytic helix.
00:42:29.08	When you mutate that, you get the following result.
00:42:33.00	So we call it, by the way, the Asn-Thumb to make a difference to the Arginine Finger.
00:42:39.03	So it's an Asn-Thumb and you'll see in a minute why we call it an Asn-Thumb.
00:42:44.26	So, first of all, if you now look at the mutation that I talked about,
00:42:48.27	so if you take the N290A mutation and analyze the reaction by, again,
00:42:55.09	this fluorescent stopped-flow assay, you see that while wild type makes a complex
00:43:00.25	and then decays into product,
00:43:02.26	the mutation here makes a complex, this is even, by the way, tighter than in the wild type case,
00:43:09.15	but there's absolutely no hydrolysis.
00:43:11.07	The reaction goes on and on. It stays up there and never goes down.
00:43:14.21	Whereas if you make a mutation like H287 to alanine,
00:43:20.22	you see that there is no complex formation because the fluorescence stays down here.
00:43:24.04	So that reaction is dead because it cannot bind
00:43:27.08	but the red reaction is deal because we think that this is the important catalytic residue.
00:43:32.12	We think that from looking just at the biochemistry.
00:43:35.15	Obviously, to know what it does, we need again to solve the structure,
00:43:38.26	which we did. This is the Rap-RapGAP complex.
00:43:43.15	And you see here the red and the green stuff is RapGAP and the blue stuff is Rap.
00:43:49.20	And you see GTP. But what you also see if you look at it in detail,
00:43:54.02	is that there is, again, something pointing from the red catalytic domain of RapGAP
00:43:59.06	into the active site and that is an asparagine.
00:44:01.17	Obviously, it's the asparagine that I've talked about.
00:44:04.20	which pokes into the active site of Rap. That's why we call it the Asn-Thumb
00:44:08.28	in relation to the Arginine Finger in the other systems.
00:44:12.27	And if you look in detail at what happens at the active site,
00:44:17.02	and if you compare it to three other structures of Ras protein and their cognate GAPs,
00:44:26.19	taking Ran and RanGAP, Ras and RasGAP, and Rho and RhoGAP,
00:44:31.18	where Rho and RhoGAP are from Rittinger et al. and the other structures are from us,
00:44:35.14	you see that the other three, these three structures here,
00:44:40.12	have a glutamine pointing towards the catalytic water,
00:44:44.29	which attacks the gamma phosphate indicated here,
00:44:47.09	which is the aluminum fluoride,
00:44:50.01	a transition state which mimics the gamma phosphate that is transferred.
00:44:54.08	And the red structure here has, instead of the glutamine 61, has a threonine
00:45:00.19	which is pointing aways from the active site; has nothing to do with catalysis.
00:45:04.01	Instead, what you see is that the purple helix here inserts this asparagine
00:45:09.19	into the active site just exactly at the position where the others have the glutamine.
00:45:14.23	So the differences here: the three other structures have a glutamine, which is in cis
00:45:19.29	and Rap and RapGAP have an asparagine in trans
00:45:23.17	which does the same thing; namely, stabilizing the catalytic water.
00:45:30.04	And if you look at the surface of the protein,
00:45:34.01	this is Rap surface shown as a surface representation,
00:45:39.07	and added to it is just the helix from RapGAP.
00:45:43.09	It sits on the surface and inserts this asparagine into the active site.
00:45:48.13	So the gamma phosphate peaks out of that hole.
00:45:50.08	And all RapGAP really does is it has on the helix this Asn, it puts it into the active site,
00:45:57.17	and that alone seems to be, at least in a chemical sense,
00:46:03.03	responsible for stimulating the GTPase reaction by 10^5 fold
00:46:07.10	because if you mutate that thing, it still binds alright, but there's absolutely no hydrolysis.
00:46:11.22	So that makes us think about, again, the future of designing anti-Ras drugs.
00:46:18.02	If all it takes is to insert such a residue into the active site,
00:46:22.11	from a chemistry point of view it should be doable,
00:46:24.24	but we obviously have to develop molecules that bind in a correct way
00:46:28.12	onto the surface, which is not so easy and we are still thinking that we might be able to do it.
00:46:35.15	This is, again, coming back to that.
00:46:38.00	So as an approach to anti-cancer drug target,
00:46:42.03	let's find molecules that induce hydrolysis of oncogenic Ras.
00:46:46.20	And from what we have observed with RapGAP we think, we have hope that it can be done.
00:46:54.26	And the third reason why working with the Rap-RapGAP system is that
00:47:03.05	it's connected to a disease called Tuberous Sclerosis, a benign tumor.
00:47:06.19	People come in with hamartomas in many organs.
00:47:09.16	But the most obvious feature and what the name comes from is that
00:47:14.07	people have, when they do an NMR of the brain, they have
00:47:16.29	these sclerotic, tuberous sort of things in the brain that you see in the NMR of the brain.
00:47:24.17	The people have mutation in two proteins called Tuberin and Hamartin.
00:47:30.22	And Tuberin, as I showed you before, has high homology
00:47:34.22	to RapGAP in this dark blue region, this means the catalytic region.
00:47:39.07	And if you now look, for example, where patient mutations in Tuberous Sclerosis,
00:47:44.23	in Tuberin, where they are occurring.
00:47:47.00	And this is an alignment of different RapGAP sequences and they align with Tuberin sequence.
00:47:53.27	You see that most highly conserved region is, again,
00:47:57.09	this thing here (the red stuff) where you have the catalytic helix.
00:48:01.08	And one of the mutations in a patient with Tuberous Sclerosis
00:48:04.22	(and many actually have that mutation) is on
00:48:07.06	this asparagine that we know is the important catalytic residue.
00:48:11.08	So this is another version of what we have seen before;
00:48:14.21	an important catalytic residue is mutated in the disease Tuberous Sclerosis.
00:48:21.19	So let me, in the last five minutes, just give you another disease,
00:48:25.22	just to make sure that it's not just the Ras and Rap system,
00:48:28.28	that there are many diseases where the inability to hydrolyze GTP leads to very many different diseases.
00:48:35.07	So this is Retinitis pigmentosa.
00:48:38.28	People have pigments on the retina. That's what the name comes from.
00:48:42.28	And they lose vision, they lose peripheral vision.
00:48:47.00	So this is a normal person seeing that building and this is a patient with the disease
00:48:52.15	that loses more and more, progressively, his peripheral vision.
00:48:56.00	And then loses complete vision after the disease develops fully.
00:49:01.19	And there are a number of genes mutated in Retinitis pigmentosa
00:49:06.12	that are called RP12x and whatever.
00:49:09.28	And there is one form that is called RP2.
00:49:13.03	It's an X-linked disease where people have mutations in...a lot of point mutations in a protein.
00:49:21.16	We determined the structure of the protein, which looks like this.
00:49:24.24	This is a beta helix domain, and this is another domain.
00:49:27.20	And you see many of the mutations that you then analyze.
00:49:31.06	You find out that what you see is that most of the mutations
00:49:37.16	determine or mess up, probably, the structure because they are inside
00:49:41.24	the hydrophobic core of the protein and you would imagine that
00:49:45.04	they don't do anything to the catalysis or the interaction of this protein.
00:49:50.19	But there are a few mutations, indicated here, for example,
00:49:54.01	E138G or R118 to H, K, or C. So these residues point into solution.
00:50:03.27	So you would think that they do something important for interaction of the protein
00:50:08.20	or in something--that they are involved in what these proteins are normally doing.
00:50:14.20	So we solved, again, the structure of the complex between Arl3.
00:50:19.13	So we knew that RP2 interacts with Arl3, another Ras-like protein.
00:50:24.26	It's called Arl because it's an Arf Related Protein.
00:50:28.04	And we solved the structure of that. So you see this stuff here would be the RP2 in purple.
00:50:37.25	And you see in green would be Arl3 in a GTP bound form.
00:50:41.25	And what you see then, if you, again, look very closely,
00:50:44.26	you see that there is a residue from RP2, right here, which points into the active site of the Arl.
00:50:52.10	So one didn't know what RP2 was doing, but when we solved the structure,
00:50:55.17	we could immediately see that it smells like a GAP because
00:50:59.18	it puts an Arginine Finger into the active site of the other protein.
00:51:05.08	And if you look at the active site in detail you see that this would be GDP, again,
00:51:10.28	this is aluminum fluoride, this is glutamine from Arl3,
00:51:15.14	and these are three residues from RP2--Q116, R118, E138.
00:51:21.26	And, obviously, all of these residues are mutated in retinitis pigmentosa.
00:51:27.15	And you can see that the arginine is doing the same thing that you've seen now many times before.
00:51:32.22	And it is stabilized by these other residues and all three residues, when mutated,
00:51:38.10	mess up the GAP activity of RP2.
00:51:41.26	So here, the structure really told us what the protein is doing.
00:51:44.19	There was no idea about the function and the structure told us, exactly, that this is a GAP for Arl3.
00:51:53.17	So let me now come to the conclusions from what I have been telling you about.
00:52:00.12	First of all, maybe, conclusions about Ras itself because it's the most important oncogene.
00:52:06.14	It's an incomplete enzyme, it cannot hydrolyze GTP very fast.
00:52:11.09	But then comes RasGAP which stabilizes switch II and the glutamine 61,
00:52:16.10	which is the important structural catalytic element and it also supplies an arginine finger into the active site.
00:52:23.04	You have Gln1 mutation--any mutation of glutamine 61 is an oncogene
00:52:28.29	because it misses, then, the catalytic residue (the system).
00:52:33.00	You have glycine mutants which are sterically compromised to do GTP hydrolysis.
00:52:38.21	There is no way that the arginine finger
00:52:42.06	can go into its proper position when there is a mutation of glycine 12.
00:52:46.04	I also showed you that there is a strongly bound GDP-Pi intermediate.
00:52:50.02	And that Pi release in the system becomes rate-limiting.
00:52:54.28	Whereas without GAP, the chemical cleavage reaction is the rate-limiting step.
00:53:00.23	The second conclusion--more general to the Ras superfamily.
00:53:05.01	Ras proteins are all incomplete enzymes.
00:53:07.20	They all hydrolyze GTP very, very slowly.
00:53:09.27	They have cognate GAPs.
00:53:12.02	So each of the sub-family proteins and sometimes even proteins within the sub-family,
00:53:16.24	have a specific cognate GAP that are required for fast GTP hydrolysis, for catalysis.
00:53:22.17	Some GAPs supply an arginine finger
00:53:25.02	and I have shown you now many different examples--Ras and Ran and Rho.
00:53:30.15	RabGAPs deliver an agrinine and a Gln.
00:53:34.23	Some GAPs supply an Asn thumb.
00:53:37.00	I showed you the example of RapGAP and Tuberin probably does the same thing.
00:53:41.03	Pi release is very often rate-limiting.
00:53:44.10	And what is even more important, and that is my message for the whole talk,
00:53:47.19	is that the perturbed GTPase reaction is involved in a number of diseases:
00:53:53.24	cancer, neurofibromatosis, tuberous sclerosis,
00:53:58.03	retinitis pigmentosa and many more that I haven't talked about.
00:54:01.07	Thank you for your attention. But I would...
00:54:03.00	Before I stop, let me first thank the people that have done the work.
00:54:07.18	The oldest story that I have told you about is that of Ras and RasGAP,
00:54:11.22	which was done by three post-docs in the lab, Reza Ahmadian, Klaus Scheffzek and Robert Mittal.
00:54:17.23	The RapGAP story was done by the students Oli Daumke, Astrid Kramer,
00:54:23.14	Partha Chakrabarti and Andrea Scrima.
00:54:26.18	And the RP2 story was done by Stefan Veltel and Karin Kuhnel.
00:54:32.04	And a lot of the movies that I have been showing you are doen by Ingrid Vetter
00:54:36.20	who also runs my crystallography lab.
00:54:38.15	And she was very, very helpful in almost all the projects.
00:54:42.13	And we have...on the FTIR we have collaborations with
00:54:46.03	our colleagues at the University of Bochum which is Klaus Gerwert and Carsten Kotting.
00:54:51.03	Thank you for your attention.
00:54:52.10