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Session 4: An Introduction to Polyketide Assembly Lines

Transcript of Part 3: Vectorial Specificity of Assembly Lines

00:00:08.03	Hello again.
00:00:09.20	I'm Chaitan Khosla
00:00:11.11	from Stanford University,
00:00:13.10	and welcome to the third part
00:00:16.24	of this trilogy of lectures
00:00:18.29	on polyketide antibiotic biosynthesis
00:00:22.05	and assembly lines.
00:00:25.29	So, in the first lecture
00:00:27.21	I introduced you to the basics
00:00:30.02	of what an assembly line is,
00:00:33.13	what's the chemistry that happens on an assembly line,
00:00:37.03	and what an assembly line looks like.
00:00:41.15	We looked at the basic architecture
00:00:45.06	of the 6-Deoxyerythronolide B synthase,
00:00:48.19	or DEBS,
00:00:50.18	which I have used subsequently
00:00:52.27	to make most of the points
00:00:55.08	that I have made.
00:00:57.12	I introduced to you
00:00:59.16	the architecture of this assembly line,
00:01:01.25	or at least as best as we can gauge today
00:01:05.12	based on data we have.
00:01:07.24	In the second lecture
00:01:10.14	I introduced you to the tools,
00:01:13.09	and how we've used those tools
00:01:15.24	to study different kinds of questions.
00:01:18.23	So, we talked about refactoring
00:01:21.18	a metabolic pathway
00:01:23.24	like this assembly line of DEBS
00:01:26.03	in E. coli.
00:01:27.09	We talked about how to reconstitute that pathway
00:01:30.07	in a test tube.
00:01:32.02	I pointed out that you can
00:01:33.25	isolate individual reactions
00:01:36.12	on the assembly line
00:01:38.05	and look at the kinetics,
00:01:39.22	whether you're looking at extender unit selection,
00:01:41.24	translocation,
00:01:43.08	elongation,
00:01:44.27	modification events.
00:01:46.24	We also talked about how these tools
00:01:48.23	can be used to study specificity
00:01:51.03	and, in particular,
00:01:52.29	we talked about two types of specificities:
00:01:56.00	stereospecificity,
00:01:58.24	I mentioned that stereochemistry
00:02:01.06	is controlled by reductive domains,
00:02:03.23	as well as extender unit specificity,
00:02:07.17	and I mentioned, over there,
00:02:09.25	you not only have enzyme-substrate recognition
00:02:13.15	but also protein-protein recognition.
00:02:18.04	This final lecture focuses on what, in my mind,
00:02:22.10	is the grandest challenge of all
00:02:25.03	in this field:
00:02:27.05	understanding how an assembly line
00:02:30.17	actually comes about.
00:02:33.20	What do I mean by that?
00:02:37.05	I've been showing you,
00:02:39.06	quite freely I might add,
00:02:41.00	schemes like this
00:02:43.08	throughout my earlier two lectures.
00:02:47.20	Every time I show you one of these schemes
00:02:50.08	or, for that matter,
00:02:52.04	you could open your favorite journal of biochemistry today,
00:02:55.26	and chances are in any given issue,
00:02:58.07	you'll find somebody having published
00:03:00.14	yet another one of these
00:03:02.13	kinds of assembly lines
00:03:04.04	that they have cloned, sequenced,
00:03:05.21	and started to characterize.
00:03:07.07	And in all cases you'll find
00:03:09.01	cartoons like the kind you see in this scheme.
00:03:15.26	The question that lies ahead of us is,
00:03:18.11	how do we know that
00:03:21.02	this is how the assembly line works?
00:03:26.27	And one way to recognize
00:03:29.12	why that question
00:03:31.25	is not an obvious question
00:03:34.15	is by thinking back
00:03:36.08	to all those orphan polyketide assembly lines
00:03:39.23	that I talked about to you
00:03:41.27	at the beginning of my first lecture.
00:03:43.29	So, I mentioned to you,
00:03:45.29	there's these large numbers of orphan assembly lines
00:03:49.03	that make polyketides
00:03:51.10	whose structure we don't know.
00:03:54.14	If I gave you the sequence
00:03:57.02	of one of those assembly lines
00:03:59.17	and asked you
00:04:01.17	to draw me an equivalent scheme
00:04:03.06	like the kind you see in front of you
00:04:05.02	in this slide,
00:04:07.10	chances are you won't be able to do it
00:04:09.15	with any confidence.
00:04:11.26	And the reason why I can show
00:04:15.07	this scheme in this slide,
00:04:16.24	and hold my head up high,
00:04:19.03	is because I know the answer
00:04:23.07	for this assembly line.
00:04:25.26	I know the structure
00:04:27.20	of 6-Deoxyerythronolide B,
00:04:30.13	and using that knowledge I am essentially
00:04:34.14	back-programming on this assembly line
00:04:37.08	what must happen at each stage
00:04:40.07	in order to get that final product.
00:04:43.20	But that begs the question,
00:04:45.27	how does nature know
00:04:48.01	that this is how the assembly line must work?
00:04:50.27	Why does nature start here,
00:04:54.01	and move here?
00:04:55.29	Or putting it differently,
00:04:57.22	why can't nature take a product
00:05:00.09	from this module
00:05:02.09	and instead of passing it on to this module,
00:05:05.12	why can't it just skip these modules altogether
00:05:08.21	and pass it on to this?
00:05:10.29	Or, why can't it take this precursor
00:05:15.13	and pass it to some other polyketide synthase
00:05:18.05	that's floating around in the same cell?
00:05:21.20	Where are the rules
00:05:24.17	that dictate how this vectorial translocation
00:05:28.17	of polyketides
00:05:30.15	must occur in the manner
00:05:32.11	that I tell you it's occurring
00:05:34.20	in this scheme?
00:05:36.20	That is the fundamental challenge
00:05:39.21	associated with this field.
00:05:43.27	The way we think about this challenge
00:05:46.23	goes back to the work
00:05:50.08	of an inspirational enzymologist
00:05:54.26	from a previous generation,
00:05:56.16	whose name you may have heard:
00:05:58.25	Bill Jencks, or William P. Jencks.
00:06:02.29	Jencks made huge contributions
00:06:06.07	to our understanding
00:06:08.08	of how enzymes work.
00:06:10.15	One of the problems he talked about
00:06:12.29	and gave a lot of thought to,
00:06:15.09	as is discussed in this article
00:06:17.07	that I would encourage any student
00:06:19.08	who's interested in machines in biology
00:06:24.03	to read,
00:06:25.21	was the question of how do you get
00:06:28.24	vectorial processes occurring in nature,
00:06:32.19	off the back of proteins like enzymes?
00:06:37.13	This article is chock-full
00:06:40.05	of many really interesting insights
00:06:42.21	that were way ahead of their time.
00:06:46.01	I certainly don't have the time
00:06:47.27	to go into all of them.
00:06:50.03	But there's one insight
00:06:51.27	that I'm going to focus on
00:06:53.21	because I think it does a very nice job
00:06:55.27	of framing the understanding
00:06:58.11	of the vectorial chemistry
00:07:00.05	that I'm here to tell you about.
00:07:02.26	What Jencks says in this article
00:07:07.03	is that proteins that do coupled vectorial processes,
00:07:10.27	that do vectorial processes,
00:07:13.06	differ from your garden variety enzyme
00:07:16.13	in the sense that they undergo large changes
00:07:21.08	in substrate specificity
00:07:23.14	at different points in their overall catalytic cycle.
00:07:29.26	And a really good example
00:07:32.14	that he uses in his quintessential
00:07:35.01	chicken-scratch cartoon manner
00:07:37.27	that's shown over here
00:07:40.08	is the example of how
00:07:43.03	myosin moves on actin fibers.
00:07:46.15	That you probably know about
00:07:49.00	because it's what textbooks are made up...
00:07:51.09	biochemistry textbooks are made of.
00:07:53.18	What you know is that myosin
00:07:56.07	moves along an actin highway
00:07:59.16	that is made up of actin monomers
00:08:02.15	by essentially going from one monomer of actin
00:08:07.15	to the next and so on.
00:08:11.07	And that movement
00:08:13.18	is powered by the hydrolysis of ATP,
00:08:17.19	by myosin.
00:08:19.28	And what Jencks postulated back then,
00:08:22.14	or what he articulated in this paper,
00:08:25.28	was that the reason you get this vectorial movement
00:08:32.07	is because there are different states of myosin
00:08:35.15	at different points in the overall cycle,
00:08:39.15	and some of these states
00:08:42.07	bind tightly to myosin,
00:08:44.22	the unbound states,
00:08:47.03	whereas ATP- or ADP-bound states,
00:08:50.19	the myosin does not bind very tightly
00:08:54.00	to actin.
00:08:56.21	The key point that Jencks made
00:08:59.14	is the reason this is a machine,
00:09:01.22	and it's not like myosin
00:09:03.23	dances back and forth on this actin highway,
00:09:07.09	the reason it motors along this actin highway
00:09:10.25	is because there is a distinct change
00:09:14.27	in specificity of myosin for actin
00:09:18.14	at different points in the catalytic cycle.
00:09:21.21	When myosin binds to ATP,
00:09:26.01	it exchanges with an unbound state
00:09:32.03	that binds to one monomer of actin,
00:09:36.02	whereas when that ATP is hydrolyzed,
00:09:39.23	that myosin sets up the binding
00:09:44.09	of a distinct conformational state
00:09:46.29	to the next.
00:09:49.03	It can't...
00:09:51.11	this exchange between the bound and the unbound state
00:09:54.29	is prohibited, because if this happened
00:09:57.03	you would be wasting this ATP hydrolysis,
00:10:00.21	and you'd be back to where you started from.
00:10:04.01	But the reason this moves forward
00:10:06.20	is because this state
00:10:09.00	has a distinct specificity
00:10:12.23	than this state of the enzyme.
00:10:15.27	So the big changes
00:10:19.02	in the specificity of myosin
00:10:21.13	are what Jencks proposed
00:10:23.12	is the basis for this vectorial process.
00:10:28.02	We think about our challenge
00:10:32.06	very much analogous
00:10:34.17	to how Jencks proposed
00:10:37.13	how myosin works on actin.
00:10:39.29	And more specifically,
00:10:42.10	there are two postulates
00:10:44.20	that we draw upon
00:10:46.28	from Jencks' analogy
00:10:49.05	to actin and myosin
00:10:51.07	to explain how this vectorial process happens.
00:10:55.08	The first one
00:10:57.19	is that the specificity change
00:11:00.14	that Jencks talked about,
00:11:03.05	in our context,
00:11:05.23	happens at the level of protein-protein interactions.
00:11:09.26	I gave you an example
00:11:12.04	in my previous lecture
00:11:14.04	of an acyltransferase
00:11:16.21	that has protein-protein specificity
00:11:19.14	in addition to its specificity
00:11:21.24	for its normal substrate.
00:11:23.18	I'll be elaborating on this theme
00:11:25.19	much more in this lecture.
00:11:28.07	And the second hypothesis
00:11:31.09	that helps us think about assembly line
00:11:34.20	vectorial biochemistry
00:11:37.03	is that this core of the protein
00:11:39.22	that exists in each module,
00:11:41.20	this KS and AT,
00:11:44.26	has specificity for the acyl carrier protein,
00:11:50.15	in an interesting way,
00:11:52.20	that leads to establishing directionality.
00:11:55.26	Bear with me if that's not clear.
00:11:58.10	Let me first start by giving you
00:12:00.14	some information, some background
00:12:02.18	on the relevance of protein-protein interactions.
00:12:05.17	So, about 15 years ago,
00:12:09.07	when Rajesh Gokhale was in my lab,
00:12:12.08	he came up with an interesting postulate
00:12:15.27	about how assembly line directionality happens.
00:12:21.02	He hypothesized
00:12:22.27	that specificity that leads to this chain
00:12:26.07	moving from this module to this module
00:12:29.08	is not in the active sites
00:12:32.12	for different substrates,
00:12:35.04	but lies at the interface
00:12:37.18	between these modules.
00:12:39.22	So you have these docking sites
00:12:43.08	that exist between modules,
00:12:46.02	that are orthogonal
00:12:48.13	to the core modules
00:12:50.22	that are doing the actual enzymology,
00:12:53.19	that allow two modules
00:12:56.05	to come together
00:12:58.04	so that you have this directionality
00:13:01.05	that is established.
00:13:03.03	How do we know that?
00:13:04.28	We know that through experiments like this.
00:13:07.19	This is one example of an experiment
00:13:09.14	that Stuart Tsuji did in my lab
00:13:11.26	some years ago
00:13:14.08	where he kinetically isolated,
00:13:16.25	from the entire DEBS assembly line
00:13:19.28	you see over here,
00:13:22.03	these two modules,
00:13:24.00	Module 2 and Module 3.
00:13:25.21	He pulled them out of the overall system,
00:13:28.09	put them into a test tube,
00:13:30.09	and reconstituted their full enzymology
00:13:33.16	that's shown in this scheme,
00:13:36.06	measuring the appropriate parameters
00:13:38.21	that one gets from these kinds of experiments.
00:13:43.05	Now, what he does is
00:13:45.19	he switches these short peptide linkers
00:13:48.11	that flank these modules
00:13:50.25	for their counterparts from this module.
00:13:54.29	And if he switches just one of the two linkers,
00:13:58.25	what he gets are two modules
00:14:01.18	that by themselves
00:14:03.25	are just as good of catalysts as they were here,
00:14:08.02	but when you put them together,
00:14:10.04	they are simply unable to talk to each other.
00:14:15.07	If, however,
00:14:16.26	you take this module that has this linker
00:14:19.08	instead of this linker,
00:14:21.25	and couple it to this module
00:14:26.23	that has this linker
00:14:28.29	instead of this linker,
00:14:31.12	this pair over here
00:14:33.18	is indistinguishable from this pair.
00:14:36.23	And so now you see
00:14:39.06	how these matching docking sites
00:14:43.22	provide for the ability for Module 2
00:14:47.02	to talk to Module 3,
00:14:49.20	as opposed to Module 5
00:14:51.23	or any of the other modules.
00:14:53.25	So you're seeing the establishing of directionality
00:14:57.14	off the back of protein-protein interactions.
00:15:02.27	Now, what the Gokhale/Tsuji experiments do
00:15:05.18	is they tell us why Module talks to Module 3.
00:15:09.21	What they don't tell us is,
00:15:12.01	why don't chains go back and forth?
00:15:15.00	It's analogous to the question
00:15:17.09	that Jencks was asking:
00:15:18.28	why is it that myosin moves
00:15:20.21	in only one direction on actin,
00:15:23.12	as opposed to just going back and forth,
00:15:25.19	back and forth?
00:15:27.20	That question has an exact analogy over here,
00:15:30.27	because as you'll remember from the first lecture
00:15:33.11	of my three lectures,
00:15:35.06	I introduced to you that chain translocation
00:15:38.09	-- when a chain moves from one module
00:15:40.02	to the next --
00:15:41.24	it does so by a thiol
00:15:43.27	to a thioester exchange reaction.
00:15:47.05	And that reaction
00:15:48.26	can just as easily run in the backward direction
00:15:51.03	as it can run in the forward direction.
00:15:53.23	And so what precludes chains
00:15:55.22	from just going back and forth
00:15:57.15	and messing up the chemistry
00:15:59.06	so you'll never get an antibiotic
00:16:00.24	out of these assembly lines?
00:16:02.22	What's written in this code
00:16:04.17	that allows that to happen?
00:16:09.05	So, the question is,
00:16:12.06	how does this event
00:16:14.25	lead to a movement here,
00:16:17.01	but when it comes time to move the chain forward,
00:16:20.14	it doesn't just go back
00:16:22.15	into the same module,
00:16:24.09	but rather goes to the next module.
00:16:29.12	Again, over here,
00:16:31.17	protein-protein interactions
00:16:33.19	play a very important role.
00:16:35.13	And in particular,
00:16:37.24	the interactions,
00:16:40.04	not just in these docking sites
00:16:42.00	that Gokhale and Tsuji identified,
00:16:43.29	but also between this protein
00:16:46.22	and this protein,
00:16:48.21	play a very critical role.
00:16:50.23	How do we know that?
00:16:52.17	Through experiments like this
00:16:54.11	that Nick Wu did,
00:16:56.06	where he isolated, kinetically,
00:16:58.02	this chain translocation event,
00:17:01.26	exposed this module over here
00:17:06.08	to the same substrate,
00:17:08.07	same docking partners,
00:17:10.15	same receiving module,
00:17:12.22	but now he changes
00:17:14.19	the identity of the donor carrier protein.
00:17:17.20	So all that's being changed over here
00:17:19.19	is the identity of this protein.
00:17:22.05	And what you see is
00:17:24.11	you have a big difference
00:17:26.19	in specificity constants
00:17:28.24	just based upon who this player is
00:17:32.19	in the overall chain translocation event,
00:17:35.11	suggesting that these protein-protein interactions
00:17:39.08	are very important.
00:17:41.17	We now know,
00:17:43.06	through work of Shiven Kapur
00:17:45.07	and others in my lab,
00:17:47.01	what the basis for this is.
00:17:49.11	So, it turns out that these carrier proteins,
00:17:51.29	which are helical proteins,
00:17:54.11	have different helices
00:17:56.10	-- different portions of this helical protein --
00:17:59.12	that dictate specificity at different events.
00:18:02.22	How do we know that?
00:18:05.02	So, you can take this red carrier protein
00:18:07.23	that talks to this red acceptor module,
00:18:12.06	replace it with the green carrier protein,
00:18:15.05	it doesn't do a good job of talking
00:18:17.14	to this acceptor module,
00:18:19.12	but now if you just put a portion of this helix
00:18:22.14	into this protein,
00:18:24.14	now this chimeric protein
00:18:26.19	is just as good,
00:18:28.12	or almost just as good,
00:18:30.06	as this red carrier protein
00:18:32.23	in delivering cargo
00:18:34.21	through this chain translocation event,
00:18:37.10	suggesting that it's this helix over here
00:18:41.02	that plays a very important role
00:18:43.08	in dictating the specificity
00:18:45.02	during chain translocation.
00:18:47.28	You can do exactly analogous experiments
00:18:50.05	in that other theoretical reaction,
00:18:53.22	the chain elongation reaction
00:18:55.23	where that carbon-carbon bond is formed
00:18:58.00	between the extender unit and the growing polyketide chain
00:19:01.23	to give you this newly elongated chain.
00:19:04.25	You can kinetically isolate this event,
00:19:07.10	you can use the same substrates,
00:19:10.20	same catalysts,
00:19:12.28	just change the identity of this carrier protein.
00:19:16.06	And what you see is,
00:19:17.28	depending on which module you use,
00:19:19.24	you can have big effects
00:19:21.23	on the specificity constant over here,
00:19:24.11	as Alice Chen showed in these experiments.
00:19:27.22	You can use the same kind of approaches
00:19:30.10	that Shiven Kapur used
00:19:32.05	to make chimeric carrier proteins
00:19:34.11	and show which portions
00:19:36.16	of these carrier proteins
00:19:38.12	play a critical role in dictating the specificity
00:19:42.22	that is measured in these parameters.
00:19:45.11	And so the take-home message
00:19:47.23	from experiments like this
00:19:51.03	is that even though chain elongation
00:19:55.07	and chain translocation
00:19:57.19	are sequential events
00:20:00.19	along the assembly line,
00:20:02.16	and even though all of those events
00:20:04.24	involve ketosynthase-acyl carrier protein interactions,
00:20:09.14	the specificity of those events
00:20:12.04	is being driven by different faces
00:20:14.23	at this carrier protein.
00:20:17.21	So this blue face of this carrier protein
00:20:20.26	is what's driving the translocation reaction,
00:20:24.17	whereas the yellow face of the carrier protein
00:20:27.19	is what's driving the elongation reaction.
00:20:31.20	So you have this Janus-like biochemistry
00:20:36.01	in these carrier proteins.
00:20:38.19	And the reason that allows you to do things
00:20:42.19	in a vectorial manner
00:20:45.13	is because now you can set up
00:20:48.18	so that a carrier protein
00:20:51.06	can only recognize the next module.
00:20:54.06	How do we know that?
00:20:56.01	We know that through experiments
00:20:57.23	where we engineer these carrier proteins
00:21:00.06	to be confused carrier proteins.
00:21:03.07	This is one example of that experiment,
00:21:05.16	where we engineer a module
00:21:07.29	that stutters.
00:21:10.09	So if you take a natural module,
00:21:12.12	like this one shown over here,
00:21:14.09	and its own carrier protein over here,
00:21:16.18	you give it a nucleophilic substrate
00:21:18.23	and electrophilic growing polyketide chain,
00:21:22.10	and you ask it to catalyze chain elongation,
00:21:25.00	it'll do so,
00:21:27.06	it'll do one round of chain elongation,
00:21:31.01	and it'll stop at this point.
00:21:34.07	And the reason it stops at this point
00:21:36.28	and doesn't go back
00:21:39.09	to the same carrier protein
00:21:41.08	is that this carrier protein
00:21:43.27	is a partner of this ketosynthase
00:21:46.17	in this chain elongation reaction
00:21:49.10	that forms this critical carbon-carbon bond,
00:21:52.22	but it is not a partner
00:21:55.16	for chain translocation.
00:21:57.16	Its chain translocation partner
00:21:59.13	is elsewhere on that assembly line.
00:22:02.14	And so the way you can test that
00:22:05.22	is you can take the recognition features
00:22:08.14	of the next carrier protein,
00:22:11.29	build them into this system
00:22:14.16	to generate a chimeric carrier protein
00:22:17.26	that can not only do a chain elongation event,
00:22:21.13	but this chimeric thing
00:22:23.12	can then donate it back
00:22:25.10	to this ketosynthase
00:22:27.19	and go through yet another round of chain elongation
00:22:30.11	to give you a longer module.
00:22:32.15	So here you've got a module
00:22:34.19	that is stuttering;
00:22:37.18	that's doing two rounds of elongation
00:22:40.07	instead of one and only one round,
00:22:42.10	because we've kind of scrambled up its recognition features.
00:22:47.02	And so what that allows me to do
00:22:49.10	is to leave you with a basic picture
00:22:52.17	of how you might wanna think about this assembly line.
00:22:56.12	So, I showed you this cartoon.
00:22:59.27	The question we're asking is,
00:23:01.29	how does the growing polyketide chain
00:23:05.10	pick one and only one path?
00:23:07.17	It's going through at least
00:23:09.17	300 Ångstroms of distance
00:23:11.19	as it travels through these modules
00:23:14.21	from one active site to another,
00:23:17.08	and comes out as a 6-Deoxyerythronolide B.
00:23:21.11	How does it pick one and only one path
00:23:24.08	to take through this entire assembly line?
00:23:27.27	And so, if we isolate just these two modules,
00:23:31.05	Modules 2 and 3,
00:23:33.24	I can show you what I think
00:23:36.05	might be a model for how this works.
00:23:39.18	So we start off
00:23:42.00	with the upstream module,
00:23:44.12	this pink module,
00:23:46.26	bound to its carrier protein
00:23:49.16	in the elongation state
00:23:51.19	-- what Jencks would call...
00:23:53.28	has specificity for the elongation state --
00:23:57.06	and that specificity
00:23:59.06	is derived from this yellow portion
00:24:01.07	of the carrier protein
00:24:03.11	that docks into this active site
00:24:05.14	and puts its substrate
00:24:07.17	where it belongs.
00:24:09.25	Meanwhile, the green carrier protein
00:24:11.24	of this green module
00:24:13.28	is just sitting in neutral mode.
00:24:16.25	So this carrier protein
00:24:19.02	then catalyzes chain elongation,
00:24:22.26	it yanks out that chain,
00:24:25.01	it can't return it back to the same site
00:24:27.05	because it doesn't have the appropriate recognition features,
00:24:30.27	instead its got recognition features, now,
00:24:34.00	using that panhandle linker,
00:24:36.06	that docking site that was identified as well as this helix
00:24:41.14	that Kapur identified.
00:24:44.02	It docks into this module,
00:24:46.11	but in a different orientation,
00:24:48.11	so now it can translocate
00:24:50.20	the chain to the next module.
00:24:54.14	When that happens,
00:24:56.13	that carrier protein goes back
00:24:58.13	into the neutral mode
00:25:00.15	and this carrier protein,
00:25:02.03	which is charged with the extender unit,
00:25:04.09	comes in to do the elongation reaction.
00:25:07.15	And so this three-stroke process
00:25:10.11	goes through again and again
00:25:12.19	to give you the final product,
00:25:15.19	to allow chains
00:25:17.13	to move forward through the assembly line.
00:25:21.10	And so I'll leave you with this picture.
00:25:24.19	I started with the picture to the left,
00:25:27.13	to motivate these remarkable antibiotic machines.
00:25:31.16	I've added one more picture
00:25:33.11	that I suspect most of you
00:25:34.26	who've had a class in biochemistry recognize.
00:25:38.10	It is the picture that we often use
00:25:40.18	to describe how proteins are made
00:25:43.06	on ribosomes.
00:25:47.24	Let me ask you the following question:
00:25:51.03	what do these pictures mean to you?
00:25:54.27	Chances are if I just flashed
00:25:57.11	both these pictures in front of you,
00:26:00.10	you would immediately recognize
00:26:02.11	what I'm saying.
00:26:04.27	If, on the other hand,
00:26:07.08	you had just arrived today from Mars,
00:26:10.08	and I showed you these pictures,
00:26:13.02	they would mean absolutely nothing to you.
00:26:16.29	So why do these pictures mean so much to you?
00:26:21.00	I would submit to you they mean a lot to you
00:26:24.03	because you have seen the movie in each case,
00:26:28.04	and that is where I will leave you
00:26:30.21	as the future of my field.
00:26:35.12	What I showed you in the past three slides
00:26:39.13	were essentially the first three frames
00:26:42.22	of a movie
00:26:45.15	that will be the DEBS movie
00:26:47.26	over the next decade.
00:26:50.09	There's no Hollywood,
00:26:51.26	no Disney as yet,
00:26:54.06	but it's backed up by
00:26:58.01	data based upon...
00:27:00.11	through a combination of biochemistry,
00:27:03.14	structural biology,
00:27:05.09	and similar types of chemical/biological approaches.
00:27:10.01	Our challenge for this field
00:27:11.27	is to take those static pictures
00:27:15.06	that I've been showing you so far
00:27:17.15	and convert them into a movie,
00:27:19.22	so once you've seen that movie,
00:27:21.29	you will recognize the engineering implications
00:27:25.01	of every state
00:27:27.07	in every one of those thousands of assembly lines
00:27:30.20	that nature has give you,
00:27:32.21	and be able to rationally think
00:27:34.23	about how to recombine them,
00:27:36.23	or how to extract interesting antibiotics
00:27:39.23	from the right kind of assembly line in nature.
00:27:43.06	I can't do that today,
00:27:46.17	nor can any of my coworkers.
00:27:49.24	But I hope this inspires you
00:27:52.16	to think about how that might be done in the future.
00:27:55.03	This brings me to the end of the lecture.
00:27:58.16	I'd like to thank you for your attention.
00:28:02.24	I'd like to thank all of my students
00:28:05.16	and postdoctoral fellows
00:28:07.21	who, over the years,
00:28:09.16	have worked with me on DEBS
00:28:11.26	and related polyketide assembly lines.
00:28:14.29	Their names show up to the left of the slide,
00:28:18.27	and I've been acknowledging their papers
00:28:21.18	as we've gone along through
00:28:23.20	these different modules.
00:28:26.27	And I'd like to also thank my collaborators:
00:28:30.20	David Cane at Brown University,
00:28:33.05	with whom I've had a long-standing collaboration
00:28:36.19	on the study of DEBS,
00:28:38.23	as well as my collaborators
00:28:41.00	at the synchrotron
00:28:42.23	-- SLAC synchrotron --
00:28:44.21	Matthews Irimpan,
00:28:46.02	Tsutomu Matsui,
00:28:47.25	and Thomas Weiss,
00:28:49.11	with whom we've had very productive collaborations
00:28:51.25	associated with the
00:28:54.05	crystallographic and SAXS characterization
00:28:56.29	of DEBS.
00:28:58.12	And last, but not least,
00:29:00.03	I'd like to thank the NIH
00:29:02.01	for having supported all our work on DEBS
00:29:05.13	over the past 15 years.
00:29:07.27	Thank you.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. MCB-1052331. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speaker and do not necessarily represent the views of iBiology, the National Science Foundation, the National Institutes of Health, or other iBiology funders.

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