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.