WEBVTT 00:00:00.500 --> 00:00:02.830 The following content is provided under a Creative 00:00:02.830 --> 00:00:04.370 Commons license. 00:00:04.370 --> 00:00:06.670 Your support will help MIT OpenCourseWare 00:00:06.670 --> 00:00:11.030 continue to offer high quality educational resources for free. 00:00:11.030 --> 00:00:13.660 To make a donation or view additional materials 00:00:13.660 --> 00:00:17.610 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.610 --> 00:00:18.520 at ocw.mit.edu. 00:00:26.040 --> 00:00:28.500 ELIZABETH NOLAN: Welcome to the class. 00:00:28.500 --> 00:00:30.990 We're going to discuss the themes that 00:00:30.990 --> 00:00:34.770 are going to basically permeate every topic 00:00:34.770 --> 00:00:37.050 and module we'll talk about here. 00:00:37.050 --> 00:00:40.140 And one of the central themes of this class 00:00:40.140 --> 00:00:41.910 is that we're interested in studying 00:00:41.910 --> 00:00:46.020 the cellular processes of life at a molecular level, right? 00:00:46.020 --> 00:00:48.240 So as biochemists and chemists, we're 00:00:48.240 --> 00:00:52.250 interested in this level of understanding. 00:00:52.250 --> 00:00:56.550 And what we see here is a cartoon depiction of the cell. 00:00:56.550 --> 00:00:59.640 And we see that there's many types of biomolecules 00:00:59.640 --> 00:01:02.800 in this environment. 00:01:02.800 --> 00:01:06.210 So what are our core themes for this year? 00:01:06.210 --> 00:01:10.890 First, we believe that life must be studied on a molecular level 00:01:10.890 --> 00:01:13.420 to truly understand it. 00:01:13.420 --> 00:01:16.990 And so we need to think about the cellular environment, 00:01:16.990 --> 00:01:21.570 both on a macroscopic scale, and on the molecular level. 00:01:21.570 --> 00:01:24.570 And this environment is complex, and it always 00:01:24.570 --> 00:01:26.100 needs to be considered, right? 00:01:26.100 --> 00:01:29.010 So as experimentalists in biochemistry, often 00:01:29.010 --> 00:01:31.680 we're doing experiments in aqueous buffer 00:01:31.680 --> 00:01:34.440 with proteins or some other biomolecule. 00:01:34.440 --> 00:01:38.160 How does that relate to a context like this one 00:01:38.160 --> 00:01:41.730 here where the environment is very different and much more 00:01:41.730 --> 00:01:44.220 complex? 00:01:44.220 --> 00:01:47.370 Something we'll see, especially in the first half, 00:01:47.370 --> 00:01:49.980 the first four modules of this course, 00:01:49.980 --> 00:01:53.730 is that in cells, complex processes 00:01:53.730 --> 00:01:55.860 are carried out by macromolecular 00:01:55.860 --> 00:01:59.160 machines and elaborate systems. 00:01:59.160 --> 00:02:01.950 And these systems are fascinating. 00:02:01.950 --> 00:02:06.854 You'll see that we know a lot, but as we learn more, 00:02:06.854 --> 00:02:08.729 there's more and more questions that come up, 00:02:08.729 --> 00:02:12.750 and more questions we need to address with that. 00:02:12.750 --> 00:02:16.140 In addition to these macromolecular machines, 00:02:16.140 --> 00:02:18.180 some additional themes for this course 00:02:18.180 --> 00:02:20.850 involve homeostasis and signaling. 00:02:20.850 --> 00:02:23.430 And these will be especially emphasized 00:02:23.430 --> 00:02:26.400 in the second half of the course when Professor Stubbe takes 00:02:26.400 --> 00:02:27.720 over there. 00:02:27.720 --> 00:02:32.610 So how do we think about homeostasis and signaling 00:02:32.610 --> 00:02:34.710 in these contexts? 00:02:34.710 --> 00:02:39.420 Something that will come up again and again 00:02:39.420 --> 00:02:43.050 is how, basically, understanding cellular processes 00:02:43.050 --> 00:02:46.350 at a molecular level, or the molecular features, 00:02:46.350 --> 00:02:49.590 can help explain mechanisms of human disease, 00:02:49.590 --> 00:02:51.510 as well as therapeutics. 00:02:51.510 --> 00:02:54.240 So an example we'll see in the early part of this lecture 00:02:54.240 --> 00:02:56.010 involves the ribosome. 00:02:56.010 --> 00:03:00.270 So many antibiotics target the ribosome. 00:03:00.270 --> 00:03:02.820 And by understanding ribosome structure and function, 00:03:02.820 --> 00:03:05.790 we can understand how these small molecule therapeutics 00:03:05.790 --> 00:03:07.080 work. 00:03:07.080 --> 00:03:11.340 Another example involves the proteasome 00:03:11.340 --> 00:03:14.890 which we'll hear about in the second half of the course. 00:03:14.890 --> 00:03:17.070 So there's therapeutics that target the proteasome, 00:03:17.070 --> 00:03:19.290 for instance, for cancer. 00:03:19.290 --> 00:03:21.580 And cholesterol biosynthesis will come up, 00:03:21.580 --> 00:03:25.080 and how does our understanding of cholesterol biosynthesis 00:03:25.080 --> 00:03:28.980 lead to ways to treat coronary disease? 00:03:28.980 --> 00:03:30.750 Something that JoAnne and I really 00:03:30.750 --> 00:03:33.120 like to think about day-to-day and convey 00:03:33.120 --> 00:03:35.580 to you in this course is the importance 00:03:35.580 --> 00:03:39.570 of experimental design, and choice of methods. 00:03:39.570 --> 00:03:42.330 So as scientists and experimentalists, 00:03:42.330 --> 00:03:45.270 how do we think about designing an experiment, 00:03:45.270 --> 00:03:48.360 because that design is really critical to the outcome, 00:03:48.360 --> 00:03:51.030 and what we can make of the data? 00:03:51.030 --> 00:03:54.630 And so throughout lectures and recitations, 00:03:54.630 --> 00:03:57.130 things to keep in mind, and that we'll reiterate, 00:03:57.130 --> 00:03:59.540 are that all techniques have inherent strengths 00:03:59.540 --> 00:04:00.875 and limitations. 00:04:00.875 --> 00:04:02.250 And so it's something we all need 00:04:02.250 --> 00:04:05.130 to keep in mind when we analyze data 00:04:05.130 --> 00:04:08.820 and think about how an experiment was done. 00:04:08.820 --> 00:04:11.850 And these systems we're going to look at in 5.08 00:04:11.850 --> 00:04:13.680 are very complex. 00:04:13.680 --> 00:04:16.529 And what that means is that many different types 00:04:16.529 --> 00:04:19.470 of experimental method are needed 00:04:19.470 --> 00:04:21.570 in order to answer complex-- 00:04:21.570 --> 00:04:23.040 and sometimes not so complex-- 00:04:23.040 --> 00:04:24.510 questions. 00:04:24.510 --> 00:04:27.310 So one method alone just often isn't enough. 00:04:27.310 --> 00:04:30.150 We need insights from many different techniques and types 00:04:30.150 --> 00:04:32.080 of expertise. 00:04:32.080 --> 00:04:36.060 And so we look forward to informing you about different 00:04:36.060 --> 00:04:39.600 types of methods-- whether they be established and quite old 00:04:39.600 --> 00:04:40.890 or new-- 00:04:40.890 --> 00:04:43.690 that are important today. 00:04:43.690 --> 00:04:46.110 And as I alluded to before, something 00:04:46.110 --> 00:04:49.980 we have to keep in mind when doing biochemistry in the lab 00:04:49.980 --> 00:04:52.650 is that the test tube is very different from the cell. 00:04:52.650 --> 00:04:55.140 These environments are vastly different, 00:04:55.140 --> 00:04:59.160 and so we always need to think about how to relate data back 00:04:59.160 --> 00:05:03.230 to a cellular or physiological context. 00:05:03.230 --> 00:05:07.860 If you measure a dissociation constant of one micromolar, 00:05:07.860 --> 00:05:11.130 what does that mean in a cell versus one picomolar, 00:05:11.130 --> 00:05:13.200 for instance. 00:05:13.200 --> 00:05:16.830 Another point to make is that the hypothesis 00:05:16.830 --> 00:05:18.660 is a moving target. 00:05:18.660 --> 00:05:22.020 So we have the hypothesis, experiments 00:05:22.020 --> 00:05:23.910 are designed to test this hypothesis, 00:05:23.910 --> 00:05:25.560 and there's some outcome. 00:05:25.560 --> 00:05:28.500 Maybe that supports the hypothesis, maybe not. 00:05:28.500 --> 00:05:30.090 Or maybe there's some new insight 00:05:30.090 --> 00:05:32.460 from a related field that really changes 00:05:32.460 --> 00:05:34.380 how we think about something. 00:05:34.380 --> 00:05:37.260 So in many cases we're integrating 00:05:37.260 --> 00:05:41.070 data and insights that are quite new, and Professor Stubbe 00:05:41.070 --> 00:05:42.930 and I won't have all of the answers. 00:05:42.930 --> 00:05:46.470 And so that type of uncertainty is something 00:05:46.470 --> 00:05:50.670 that we aim for you all to gain some level of comfort with. 00:05:50.670 --> 00:05:54.710 So there's many complexities in primary data, often 00:05:54.710 --> 00:05:55.560 uncertainties. 00:05:55.560 --> 00:05:58.530 And that's just an aspect of this course. 00:05:58.530 --> 00:06:00.780 And scientists, it's something we grapple 00:06:00.780 --> 00:06:02.920 with every day in our own work. 00:06:02.920 --> 00:06:07.050 So we're introducing that to you here. 00:06:07.050 --> 00:06:10.620 And along those lines, just keep in mind, we know so much. 00:06:10.620 --> 00:06:13.710 And I think it's amazing, and-- if I step back and think 00:06:13.710 --> 00:06:15.750 about this for some of the systems 00:06:15.750 --> 00:06:18.360 we'll see-- actually overwhelming. 00:06:18.360 --> 00:06:21.720 And it's really due to dedicated efforts of many, many people 00:06:21.720 --> 00:06:23.620 over many, many years. 00:06:23.620 --> 00:06:26.580 But with that said, there are so many remaining unanswered 00:06:26.580 --> 00:06:30.390 questions, and we hope that you'll find inspiration 00:06:30.390 --> 00:06:33.600 in some of these questions as looking forward 00:06:33.600 --> 00:06:35.260 within this field. 00:06:35.260 --> 00:06:36.980 There. 00:06:36.980 --> 00:06:42.810 OK, so what about the cell and macromolecular crowding? 00:06:42.810 --> 00:06:46.920 Just to emphasize this point a bit more, 00:06:46.920 --> 00:06:49.620 here we have an E. coli. 00:06:49.620 --> 00:06:54.630 OK, so E. coli are laboratory workhorses for biochemists. 00:06:54.630 --> 00:06:58.110 They're fascinating, I love E. coli. 00:06:58.110 --> 00:07:01.470 But I just show you this simple E. coli cartoon 00:07:01.470 --> 00:07:05.970 and this depiction here to emphasize how crowded 00:07:05.970 --> 00:07:08.460 the cellular environment is. 00:07:08.460 --> 00:07:11.490 So we have an equal E. coli of about two microns long, 00:07:11.490 --> 00:07:17.180 and maybe half a micron wide, a volume of about a femtolitre. 00:07:17.180 --> 00:07:20.130 And if we think about the E. coli genome for a minute, 00:07:20.130 --> 00:07:23.430 it encodes about 4,000 proteins. 00:07:23.430 --> 00:07:25.230 That's a lot of proteins. 00:07:25.230 --> 00:07:28.770 And if we think about one E. coli cell of this small size, 00:07:28.770 --> 00:07:33.190 can just ask a simple question, how many ribosomes are there? 00:07:33.190 --> 00:07:35.580 So we all know the ribosomes are needed 00:07:35.580 --> 00:07:38.340 for polypeptide biosynthesis. 00:07:38.340 --> 00:07:42.820 How many ribosomes are packaged in one E. coli? 00:07:42.820 --> 00:07:43.560 Any guess? 00:07:51.096 --> 00:07:54.620 So, 10, 100, a million. 00:07:54.620 --> 00:07:55.790 AUDIENCE: Order of 1,000? 00:07:55.790 --> 00:07:56.170 ELIZABETH NOLAN: Pardon? 00:07:56.170 --> 00:07:57.462 AUDIENCE: Order of like, 1,000? 00:07:57.462 --> 00:08:00.750 ELIZABETH NOLAN: Yeah, let's say 1,000 times 15 or 20. 00:08:00.750 --> 00:08:04.430 So there's about 15,000 to 20,000 ribosomes 00:08:04.430 --> 00:08:06.620 in one E. coli cell. 00:08:06.620 --> 00:08:09.740 And as we'll see in Friday's lecture, 00:08:09.740 --> 00:08:12.360 the ribosome is very large. 00:08:12.360 --> 00:08:13.520 How did they all fit? 00:08:13.520 --> 00:08:15.350 And there's not only the ribosomes, 00:08:15.350 --> 00:08:17.090 but there's many, many other players, 00:08:17.090 --> 00:08:19.315 just as noted here in this cartoon. 00:08:21.920 --> 00:08:23.930 So you can think about what does that mean 00:08:23.930 --> 00:08:25.880 in terms of concentrations. 00:08:25.880 --> 00:08:28.790 We'll bring up concentrations of biomolecules in the cell 00:08:28.790 --> 00:08:30.740 throughout this course, and what does it 00:08:30.740 --> 00:08:36.590 mean having them packaged together so much here? 00:08:36.590 --> 00:08:40.159 So, very different than the test tube. 00:08:40.159 --> 00:08:43.429 Our goals, some of which I think have 00:08:43.429 --> 00:08:46.460 been communicated by me so far. 00:08:46.460 --> 00:08:49.130 But just to emphasize, we're interested 00:08:49.130 --> 00:08:51.800 in these macromolecular machines and chemical 00:08:51.800 --> 00:08:54.770 processes responsible for life. 00:08:54.770 --> 00:08:56.480 We hope by the end of this course, 00:08:56.480 --> 00:09:00.950 everyone gains an appreciation for the complexity of life, 00:09:00.950 --> 00:09:04.730 and our current understanding of the topics 00:09:04.730 --> 00:09:07.430 we present to you this spring. 00:09:07.430 --> 00:09:10.580 There's close links between basic fundamental research 00:09:10.580 --> 00:09:15.290 and medicine, and technology development as well. 00:09:15.290 --> 00:09:18.350 Understanding the experimental basis 00:09:18.350 --> 00:09:21.710 for understanding, methods and hypotheses. 00:09:21.710 --> 00:09:25.790 And what we think is something that we hope to achieve, 00:09:25.790 --> 00:09:28.160 and that you can bring to other places 00:09:28.160 --> 00:09:31.400 after this course is really to be able to knowledgeably 00:09:31.400 --> 00:09:35.060 and critically evaluate methods and results, especially 00:09:35.060 --> 00:09:36.440 primary data. 00:09:36.440 --> 00:09:39.860 And we also hope that we convince you 00:09:39.860 --> 00:09:42.170 that biological chemistry is really 00:09:42.170 --> 00:09:44.060 thought provoking and fun, and hope 00:09:44.060 --> 00:09:47.100 you all think that right now as well. 00:09:47.100 --> 00:09:50.550 So what are the actual topics we're going to cover? 00:09:50.550 --> 00:09:53.810 We organized this course into modules, 00:09:53.810 --> 00:09:57.260 and these modules are listed here. 00:09:57.260 --> 00:10:00.170 And different modules will have different numbers 00:10:00.170 --> 00:10:02.360 of lectures dedicated to them. 00:10:02.360 --> 00:10:06.320 But where we'll go between now and spring break-- 00:10:06.320 --> 00:10:09.710 I'll present to you during these weeks-- 00:10:09.710 --> 00:10:14.300 is that we're going to focus on the lifecycle of a protein 00:10:14.300 --> 00:10:15.860 for the first three modules. 00:10:15.860 --> 00:10:18.620 And many of you are familiar with aspects of this. 00:10:18.620 --> 00:10:21.050 We're going to present these topics, 00:10:21.050 --> 00:10:23.900 I think, a bit differently than what you've seen before. 00:10:23.900 --> 00:10:26.700 Again, very much from the standpoint 00:10:26.700 --> 00:10:30.470 of experimental methods and hypothesis testing. 00:10:30.470 --> 00:10:33.200 So we'll cover protein synthesis, 00:10:33.200 --> 00:10:36.650 doing a careful case study of the ribosome. 00:10:36.650 --> 00:10:38.720 We'll continue with protein folding. 00:10:38.720 --> 00:10:41.420 So asking the question, after the ribosome 00:10:41.420 --> 00:10:45.470 synthesizes a polypeptide chain, how does that polypeptide 00:10:45.470 --> 00:10:48.500 assemble into its native form? 00:10:48.500 --> 00:10:51.500 What happens when proteins are misfolded? 00:10:51.500 --> 00:10:54.200 And then we'll move into protein degradation, 00:10:54.200 --> 00:10:58.700 and we'll look at proteases and machines that are involved 00:10:58.700 --> 00:11:01.640 in proteolytic degradation. 00:11:01.640 --> 00:11:04.160 And where we'll close the first half 00:11:04.160 --> 00:11:07.880 is with module four, which is on synthases, 00:11:07.880 --> 00:11:11.210 or often called assembly-line enzymology. 00:11:11.210 --> 00:11:14.810 And this is a different type of template-driven polymerization 00:11:14.810 --> 00:11:18.440 that's involved in the synthesis of natural products. 00:11:18.440 --> 00:11:20.570 And then after spring break, Professor Stubbe 00:11:20.570 --> 00:11:22.970 will take over, and the focus will 00:11:22.970 --> 00:11:27.080 be on cellular processes that involve homeostasis, 00:11:27.080 --> 00:11:29.330 metabolism, and signaling. 00:11:29.330 --> 00:11:34.910 And so these topics will involve cholesterol biosynthesis, 00:11:34.910 --> 00:11:38.230 and a type of molecule called terpene. 00:11:38.230 --> 00:11:41.240 And so a third way to make a carbon-carbon bond 00:11:41.240 --> 00:11:43.340 will be introduced in this section. 00:11:43.340 --> 00:11:46.220 So you've heard about Claisen and Aldol condensations 00:11:46.220 --> 00:11:50.840 in prior biochemistry courses, this will be another route. 00:11:50.840 --> 00:11:54.210 And then, we both love metals and biology, 00:11:54.210 --> 00:11:57.350 so there's a whole field of bioinorganic chemistry, 00:11:57.350 --> 00:12:01.340 and it will be introduced to you here with iron homeostasis 00:12:01.340 --> 00:12:03.980 as a case study. 00:12:03.980 --> 00:12:07.460 And moving from here, and something quite related, 00:12:07.460 --> 00:12:09.590 involves reactive oxygen species. 00:12:09.590 --> 00:12:13.460 So I'm sure you've all heard about these somewhere, maybe 00:12:13.460 --> 00:12:16.340 in the news, maybe from your lab work. 00:12:16.340 --> 00:12:19.340 What are these reactive oxygen species? 00:12:19.340 --> 00:12:20.720 Are they all reactive? 00:12:20.720 --> 00:12:23.300 What kind of chemistry do they do in a cell? 00:12:23.300 --> 00:12:26.630 How do we study that here? 00:12:26.630 --> 00:12:30.340 And then, of course, we'll close with a section, 00:12:30.340 --> 00:12:36.020 a module on nucleotide and deoxynucleotide metabolism-- 00:12:36.020 --> 00:12:38.480 excuse me-- as well as regulation. 00:12:38.480 --> 00:12:41.430 And then an integration of course concepts. 00:12:41.430 --> 00:12:44.840 So we have a lot of exciting topics and exciting things 00:12:44.840 --> 00:12:47.720 to tell you about. 00:12:47.720 --> 00:12:49.760 In terms of level of understanding 00:12:49.760 --> 00:12:54.910 for this course, as I said, many of these systems are complex. 00:12:54.910 --> 00:12:58.180 We're going to look at huge macromolecular machines, 00:12:58.180 --> 00:13:00.640 and multi-step processes. 00:13:00.640 --> 00:13:03.160 This is a biochemistry course, and we 00:13:03.160 --> 00:13:06.640 are interested in molecular level, in addition 00:13:06.640 --> 00:13:08.480 to this big picture. 00:13:08.480 --> 00:13:13.440 And so things to keep in mind when thinking about structure. 00:13:13.440 --> 00:13:15.640 You need to think about the amino acids, 00:13:15.640 --> 00:13:20.290 and please review these if you're a bit rusty. 00:13:20.290 --> 00:13:24.640 So to know the side chains, PKAs, et cetera, that's 00:13:24.640 --> 00:13:27.670 all important to have in mind. 00:13:27.670 --> 00:13:29.710 What are the protein folds? 00:13:29.710 --> 00:13:31.960 What are the arrangements of these macromolecular 00:13:31.960 --> 00:13:35.680 assemblies, and how do we study that? 00:13:35.680 --> 00:13:37.990 In terms of reactivity, we'll see 00:13:37.990 --> 00:13:40.520 bond-breaking and bond-forming reactions. 00:13:40.520 --> 00:13:44.140 So again, we need to think about things like PKAs, nucleophiles, 00:13:44.140 --> 00:13:46.060 and electrophiles. 00:13:46.060 --> 00:13:49.090 If you need to brush up, organic chemistry textbook 00:13:49.090 --> 00:13:52.800 or biochemistry textbook is a good place to go. 00:13:52.800 --> 00:13:56.410 And then something to keep in mind is dynamics. 00:13:56.410 --> 00:14:00.220 So the macromolecular structures and enzymes and proteins 00:14:00.220 --> 00:14:01.900 we'll look at are dynamic. 00:14:01.900 --> 00:14:05.140 Often we only have a static picture or some number 00:14:05.140 --> 00:14:06.910 of static pictures. 00:14:06.910 --> 00:14:09.310 But there's conformational change, 00:14:09.310 --> 00:14:11.320 transient binding occurs, and we always 00:14:11.320 --> 00:14:13.220 need to think about kinetics. 00:14:13.220 --> 00:14:15.430 So these are things just to keep in mind when you're 00:14:15.430 --> 00:14:18.700 reading and questioning to yourself about any given 00:14:18.700 --> 00:14:21.670 system here. 00:14:21.670 --> 00:14:25.570 So what about experimental methods? 00:14:25.570 --> 00:14:31.180 This is just another topic to go over in this course overview. 00:14:31.180 --> 00:14:36.010 So there's many methods that come up in 5.08. 00:14:36.010 --> 00:14:38.530 And we don't expect that you have 00:14:38.530 --> 00:14:42.700 knowledge of any or all of these at the stage of starting 00:14:42.700 --> 00:14:44.200 the course. 00:14:44.200 --> 00:14:47.350 The difficulty that comes up is that we can't introduce 00:14:47.350 --> 00:14:49.780 all of these methods to you at once 00:14:49.780 --> 00:14:54.940 in a level of detail that's needed for everything we do. 00:14:54.940 --> 00:14:57.940 OK, so what will happen is that if methods come up 00:14:57.940 --> 00:15:00.750 in problem sets that haven't yet been addressed, 00:15:00.750 --> 00:15:02.920 we'll give you enough background information 00:15:02.920 --> 00:15:05.320 in the problems that material, such that you 00:15:05.320 --> 00:15:09.700 can think about the questions and answer them. 00:15:09.700 --> 00:15:12.400 And we'll let you know when a method comes up. 00:15:12.400 --> 00:15:14.530 You know, you'll hear this in recitation x, 00:15:14.530 --> 00:15:17.560 or we'll talk about it more in class. 00:15:17.560 --> 00:15:20.110 So right now, what I'd like to do 00:15:20.110 --> 00:15:22.180 is just go over a few of the methods 00:15:22.180 --> 00:15:26.110 that you're going to see multiple times. 00:15:26.110 --> 00:15:31.660 And the thing to keep in mind is that the context in which 00:15:31.660 --> 00:15:34.690 these methods are being used may differ, 00:15:34.690 --> 00:15:39.270 but the underlying principles are the same. 00:15:39.270 --> 00:15:42.340 And we choose methods that are being used today, 00:15:42.340 --> 00:15:43.780 and are important. 00:15:43.780 --> 00:15:46.300 Some of these were developed decades ago, some of these 00:15:46.300 --> 00:15:49.480 are very, very new, and hot off the press. 00:15:49.480 --> 00:15:53.290 So if it's an older paper, please don't brush it off as, 00:15:53.290 --> 00:15:55.300 like, oh, this is old. 00:15:55.300 --> 00:15:57.940 And so, you know, it's not new. 00:15:57.940 --> 00:16:01.510 We're all really excited by technology and everything here, 00:16:01.510 --> 00:16:05.020 but many of these older methods are robust, 00:16:05.020 --> 00:16:07.630 and used all the time here. 00:16:07.630 --> 00:16:11.280 So what are some methods and tools 00:16:11.280 --> 00:16:13.030 that we'll have under our belt? 00:16:18.880 --> 00:16:21.400 The first to point out are methods involved 00:16:21.400 --> 00:16:23.050 in macromolecular structure. 00:16:34.900 --> 00:16:37.450 So we care a lot about structure, 00:16:37.450 --> 00:16:40.090 because we need structural understanding 00:16:40.090 --> 00:16:44.050 to be able to comprehend how these systems work. 00:16:44.050 --> 00:16:46.690 And so one method we'll see a lot-- 00:16:46.690 --> 00:16:49.420 and you'll discuss in recitation this week-- 00:16:49.420 --> 00:16:51.400 is x-ray crystallography. 00:16:58.270 --> 00:17:01.420 And in addition, a method that will come up quite a bit-- 00:17:01.420 --> 00:17:03.790 and we'll see both of these in the initial discussions 00:17:03.790 --> 00:17:05.500 of the ribosome-- 00:17:05.500 --> 00:17:07.050 is electron microscopy. 00:17:15.640 --> 00:17:18.990 And another method to be aware of-- and if you're curious, 00:17:18.990 --> 00:17:22.000 talk to your TA, Shiva-- 00:17:22.000 --> 00:17:23.579 is NMR. 00:17:23.579 --> 00:17:27.940 OK, so NMR has a lot of applications 00:17:27.940 --> 00:17:30.820 here within biological chemistry, 00:17:30.820 --> 00:17:33.700 but we won't discuss that. 00:17:33.700 --> 00:17:37.930 What can go along with methods is bioinformatics. 00:17:48.100 --> 00:17:51.970 So how many of you have used BLAST? 00:17:51.970 --> 00:17:53.770 How many of you know what BLAST stands for? 00:17:56.277 --> 00:17:58.110 AUDIENCE: Basic Local Alignment Search Tool. 00:17:58.110 --> 00:18:01.060 ELIZABETH NOLAN: Yeah, Basic Local Alignment Search Tool. 00:18:01.060 --> 00:18:03.960 So what does this let you do? 00:18:07.880 --> 00:18:11.510 It lets you find regions of similarity between sequences, 00:18:11.510 --> 00:18:13.970 whether that's amino acid sequence, 00:18:13.970 --> 00:18:16.140 a nucleotide sequence. 00:18:16.140 --> 00:18:20.030 And you can use that information to make hypotheses and design 00:18:20.030 --> 00:18:23.090 experiments there. 00:18:23.090 --> 00:18:24.020 So that will come up. 00:18:26.720 --> 00:18:30.710 I have additional methods and possibilities. 00:18:30.710 --> 00:18:32.090 What about fluorescence? 00:18:41.350 --> 00:18:44.890 So how many of you have done an experiment that 00:18:44.890 --> 00:18:49.360 involves fluorescence, either in lab, or in your research? 00:18:49.360 --> 00:18:53.890 How many, did that involve a fluorescent protein? 00:18:53.890 --> 00:18:57.300 What about a small molecule? 00:18:57.300 --> 00:18:59.530 Yeah. 00:18:59.530 --> 00:19:00.400 That's fluorescent. 00:19:00.400 --> 00:19:03.130 So have you thought about why the protein was 00:19:03.130 --> 00:19:07.540 used, versus maybe why a small molecule, and what 00:19:07.540 --> 00:19:09.490 are inherent strengths and limitations or one 00:19:09.490 --> 00:19:13.180 or the other, depending what you want to do? 00:19:13.180 --> 00:19:17.530 So fluorescence is used in many, many different contexts. 00:19:17.530 --> 00:19:21.410 We can think about proteins like green fluorescent protein, 00:19:21.410 --> 00:19:23.400 we can think about using small molecules. 00:19:28.750 --> 00:19:32.170 And we like fluorescence because it allows us to see. 00:19:32.170 --> 00:19:36.490 We can get visual information. 00:19:36.490 --> 00:19:39.890 And so, where fluorescence will first come up in this class 00:19:39.890 --> 00:19:42.280 is with the ribosome. 00:19:42.280 --> 00:19:44.830 And in recitation week two, there'll 00:19:44.830 --> 00:19:48.370 be some discussion about using small molecule fluorophores 00:19:48.370 --> 00:19:51.310 to label tRNAs, and using fluorescence 00:19:51.310 --> 00:19:54.980 as a readout of steps in the translation process. 00:19:54.980 --> 00:19:58.160 And there's a lot of considerations and caveats 00:19:58.160 --> 00:19:58.660 to that. 00:19:58.660 --> 00:19:59.890 Do we have a pizza delivery? 00:19:59.890 --> 00:20:01.840 Thank goodness no. 00:20:01.840 --> 00:20:04.220 Often in this class, we get pizza deliveries for someone 00:20:04.220 --> 00:20:04.720 else. 00:20:04.720 --> 00:20:07.520 I didn't know if that's already starting. 00:20:07.520 --> 00:20:09.760 Yeah, yeah. 00:20:09.760 --> 00:20:13.900 We'll also see GFP being used in the proteasome section 00:20:13.900 --> 00:20:15.080 for degradation. 00:20:15.080 --> 00:20:17.640 So a folded protein has fluorescence, 00:20:17.640 --> 00:20:21.227 a degraded protein does not. 00:20:21.227 --> 00:20:22.060 What about kinetics? 00:20:30.050 --> 00:20:35.230 So what different types of kinetic studies can be done? 00:20:35.230 --> 00:20:38.420 So what do we all hear about in introductory biochemistry 00:20:38.420 --> 00:20:38.920 class? 00:20:46.630 --> 00:20:47.570 Pardon? 00:20:47.570 --> 00:20:48.550 AUDIENCE: [INAUDIBLE]. 00:20:48.550 --> 00:20:50.717 ELIZABETH NOLAN: Yeah, steady state kinetics, right? 00:20:50.717 --> 00:20:51.830 Turnover. 00:20:51.830 --> 00:20:58.510 So we have steady state, which I encourage you to review 00:20:58.510 --> 00:21:02.080 Michaelis-Mentin Kinetics here. 00:21:02.080 --> 00:21:05.500 And you'll also be introduced in the first weeks 00:21:05.500 --> 00:21:08.500 of this course, and especially recitation three-- 00:21:08.500 --> 00:21:11.710 so recitation two is going to build up to this-- 00:21:11.710 --> 00:21:12.910 pre-steady state kinetics. 00:21:24.870 --> 00:21:29.550 So here, you've heard about this in 5.07 00:21:29.550 --> 00:21:33.030 or another course, introductory course. 00:21:33.030 --> 00:21:37.650 And we're looking at multiple turnover of an enzyme. 00:21:41.260 --> 00:21:45.260 And these experiments are set up with an excess of substrate, 00:21:45.260 --> 00:21:47.170 right, in order to afford conditions 00:21:47.170 --> 00:21:48.430 that allow multiple turnover. 00:21:57.440 --> 00:22:00.620 So there's formation of an enzyme substrate complex, 00:22:00.620 --> 00:22:02.750 and then there's product formation. 00:22:02.750 --> 00:22:04.610 So review as needed. 00:22:04.610 --> 00:22:09.230 So what about pre-steady state kinetics? 00:22:09.230 --> 00:22:13.990 How many of you are familiar with this method? 00:22:13.990 --> 00:22:14.740 Not so much. 00:22:14.740 --> 00:22:16.225 So what does the name suggest? 00:22:20.084 --> 00:22:20.584 Pardon? 00:22:25.682 --> 00:22:27.890 JOANNE STUBBE: So I'm deaf, you have to speak louder. 00:22:27.890 --> 00:22:29.680 ELIZABETH NOLAN: Yeah, we're both deaf. 00:22:29.798 --> 00:22:31.090 JOANNE STUBBE: I'm really deaf. 00:22:31.090 --> 00:22:33.310 So if you want to say something, so I can hear it. 00:22:35.880 --> 00:22:36.742 Speak up. 00:22:36.742 --> 00:22:38.700 AUDIENCE: Yeah, maybe observing single molecule 00:22:38.700 --> 00:22:40.180 by some spectroscopy. 00:22:40.180 --> 00:22:42.310 ELIZABETH NOLAN: Yeah, a single turnover, maybe, I 00:22:42.310 --> 00:22:43.560 think is what. 00:22:43.560 --> 00:22:45.460 If we're having multiple turnovers here 00:22:45.460 --> 00:22:47.380 in the steady state, right? 00:22:47.380 --> 00:22:49.150 If we're before the steady state, 00:22:49.150 --> 00:22:51.700 what does that mean, right? 00:22:51.700 --> 00:22:53.530 It means we're in the initial, really 00:22:53.530 --> 00:22:56.950 initial part of this reaction, where we're looking 00:22:56.950 --> 00:22:58.435 at a single turnover here. 00:23:02.130 --> 00:23:03.255 And how would you do this? 00:23:06.040 --> 00:23:11.610 Basically, you look with subs having limiting substrate 00:23:11.610 --> 00:23:14.880 rather than excess substrate. 00:23:14.880 --> 00:23:20.040 And this is just to give a little prelude 00:23:20.040 --> 00:23:23.850 in terms of thinking about experimental design. 00:23:23.850 --> 00:23:27.735 So here, look at the first moments of a reaction. 00:23:34.920 --> 00:23:39.168 So what type of time scale is that? 00:23:39.168 --> 00:23:40.032 AUDIENCE: Small. 00:23:40.032 --> 00:23:41.710 ELIZABETH NOLAN: Yeah, small. 00:23:41.710 --> 00:23:46.390 Maybe a millisecond time scale, compared to a timescale 00:23:46.390 --> 00:23:48.850 of seconds or minutes. 00:23:48.850 --> 00:23:50.120 So what does that mean? 00:23:50.120 --> 00:23:53.400 It means you need some different experimental setup. 00:23:53.400 --> 00:23:55.420 You can't do pre-steady state kinetics 00:23:55.420 --> 00:23:58.480 in the way we've done steady state kinetics, say in a lab 00:23:58.480 --> 00:24:00.420 class for instance. 00:24:00.420 --> 00:24:02.570 So you need a special apparatus. 00:24:02.570 --> 00:24:06.700 And what does it let you see? 00:24:06.700 --> 00:24:11.460 Here you're looking at multiple turnover, products forming. 00:24:11.460 --> 00:24:15.430 You know, here in the early stages, what can you see? 00:24:15.430 --> 00:24:18.970 Maybe intermediate formation. 00:24:18.970 --> 00:24:22.690 And why might that be important for thinking about mechanism? 00:24:22.690 --> 00:24:25.360 So those will come up in the first weeks of recitation. 00:24:28.380 --> 00:24:33.240 Another topic that will come up, and is something 00:24:33.240 --> 00:24:37.470 that you always need to think about, and relates 00:24:37.470 --> 00:24:42.360 to integrity of materials, is that of purification. 00:24:42.360 --> 00:24:45.240 So how are proteins purified. 00:24:45.240 --> 00:24:48.630 For studying the ribosome, how do we 00:24:48.630 --> 00:24:52.770 get ribosomes that are pure and are correct? 00:24:52.770 --> 00:24:55.680 Or what if you'd like to use a mutant ribosome? 00:24:55.680 --> 00:24:59.610 How does that get generated? 00:24:59.610 --> 00:25:09.390 So here, you can talk about ribosome or protein 00:25:09.390 --> 00:25:12.530 purification. 00:25:12.530 --> 00:25:17.610 And so, I'll present to you on ribosomes and mutant ribosomes 00:25:17.610 --> 00:25:19.780 in week four of recitation. 00:25:19.780 --> 00:25:23.340 And this topic more generally of proteins 00:25:23.340 --> 00:25:26.610 will come up in passing again and again. 00:25:26.610 --> 00:25:30.860 So how many of you have purified a protein? 00:25:30.860 --> 00:25:31.360 Many. 00:25:31.360 --> 00:25:33.370 How many of you used an affinity tag? 00:25:36.800 --> 00:25:38.950 So are they the answer to all problems? 00:25:41.920 --> 00:25:42.420 No. 00:25:42.420 --> 00:25:46.860 They can be a huge help, but they can also be problematic 00:25:46.860 --> 00:25:48.480 in one way or another, right? 00:25:48.480 --> 00:25:50.790 So with the ribosome we'll look at a case 00:25:50.790 --> 00:25:53.310 where there was really some elegant work done 00:25:53.310 --> 00:25:57.960 using an affinity tag approach to allow researchers 00:25:57.960 --> 00:26:00.510 to obtain new ribosomes. 00:26:00.510 --> 00:26:02.760 We'll also, though, talk about the limitations 00:26:02.760 --> 00:26:04.732 of that type of methodology, and the things 00:26:04.732 --> 00:26:06.690 you need to think about if you're doing protein 00:26:06.690 --> 00:26:11.100 biochemistry, and how a tag may affect your experiments 00:26:11.100 --> 00:26:14.370 and data there. 00:26:14.370 --> 00:26:29.470 In addition, to think about is assay development, 00:26:29.470 --> 00:26:30.580 and analytical methods. 00:26:40.460 --> 00:26:44.630 And so there will be many different types of assays that 00:26:44.630 --> 00:26:47.360 are presented in this course. 00:26:47.360 --> 00:26:49.610 And something just to think about-- 00:26:49.610 --> 00:26:55.180 how do you develop the right assay, and what 00:26:55.180 --> 00:26:59.170 are all the considerations? 00:26:59.170 --> 00:27:03.310 How do you know your assay is a good one for the question you 00:27:03.310 --> 00:27:05.500 want to address there? 00:27:05.500 --> 00:27:08.510 This is actually really complicated. 00:27:08.510 --> 00:27:11.860 And so there'll be some case studies 00:27:11.860 --> 00:27:14.050 that come up in the course, but just more broadly 00:27:14.050 --> 00:27:14.710 to think about. 00:27:14.710 --> 00:27:18.250 So often in lab classes, you may have an assay, 00:27:18.250 --> 00:27:21.550 but you might not be aware of all of the considerations that 00:27:21.550 --> 00:27:27.430 went into actually developing that assay such that it works. 00:27:27.430 --> 00:27:29.410 And then there's the analytical methods 00:27:29.410 --> 00:27:32.020 that are used, either for analyzing 00:27:32.020 --> 00:27:34.790 assay data or other data. 00:27:34.790 --> 00:27:37.540 And again, these have strengths and limitations. 00:27:37.540 --> 00:27:47.500 Just some that will come up, to present western blots 00:27:47.500 --> 00:27:49.070 and immunoprecipitation. 00:27:57.120 --> 00:27:59.670 So these methods involve antibodies, 00:27:59.670 --> 00:28:05.490 and so we need to think about the antibodies themselves here. 00:28:05.490 --> 00:28:06.660 Radioactivity. 00:28:12.670 --> 00:28:15.450 OK, how does this work? 00:28:15.450 --> 00:28:19.040 Why do biochemists like to use radioactivity and assay 00:28:19.040 --> 00:28:19.850 development? 00:28:19.850 --> 00:28:23.450 And how to think about this productively and correctly. 00:28:23.450 --> 00:28:31.160 So should you be afraid of iron-55, yes or no? 00:28:31.160 --> 00:28:34.160 How does that exposure compare to being in an airplane, 00:28:34.160 --> 00:28:36.500 for instance. 00:28:36.500 --> 00:28:39.500 Seriously, because there's a lot of fear associated 00:28:39.500 --> 00:28:42.500 with radioactivity that may or may not be well-founded, 00:28:42.500 --> 00:28:44.930 depending on what you're doing. 00:28:44.930 --> 00:28:47.840 And so this gives us a lot of sensitivity. 00:28:47.840 --> 00:28:50.750 And JoAnne will talk in week two of recitation 00:28:50.750 --> 00:28:54.170 about radioactivity, and designing experiments 00:28:54.170 --> 00:28:58.160 that use this as a read out. 00:28:58.160 --> 00:28:59.150 What else? 00:28:59.150 --> 00:29:00.740 So affinity measurements. 00:29:10.380 --> 00:29:14.580 OK, so dissociation constants, or affinity constants, 00:29:14.580 --> 00:29:17.460 how are these measured? 00:29:17.460 --> 00:29:20.790 When reading the literature, is the value a good one, 00:29:20.790 --> 00:29:25.410 or a not-so-good one, and how can you make that distinction? 00:29:25.410 --> 00:29:26.870 Mass spec and proteomics. 00:29:35.740 --> 00:29:37.950 So these will be in the later half of the class-- 00:29:37.950 --> 00:29:41.020 I believe recitations 11 and 12-- 00:29:41.020 --> 00:29:42.700 and many others. 00:29:42.700 --> 00:29:44.800 And we're introducing CRISPR this year, 00:29:44.800 --> 00:29:48.880 in the context of the cholesterol unit as well. 00:29:48.880 --> 00:29:52.120 So as I said, we can't take care of all of these methods 00:29:52.120 --> 00:29:53.670 immediately. 00:29:53.670 --> 00:29:55.420 We'll let you know when they're coming up, 00:29:55.420 --> 00:30:00.370 when you need to know more details about them as we 00:30:00.370 --> 00:30:05.320 go through the course here for that. 00:30:05.320 --> 00:30:08.560 So we can get started. 00:30:08.560 --> 00:30:11.410 And in the last few minutes, what 00:30:11.410 --> 00:30:16.900 I'll do is just give you a brief overview of the macromolecular 00:30:16.900 --> 00:30:22.390 machines we'll look at through modules one through three. 00:30:25.190 --> 00:30:28.020 And basically, what is the big picture? 00:30:28.020 --> 00:30:30.890 And then we're going to break that down 00:30:30.890 --> 00:30:34.110 into looking at individual components. 00:30:34.110 --> 00:30:44.210 So if we think about the lifecycle of a protein, 00:30:44.210 --> 00:30:49.520 basically, we'll fast forward to having mRNA from transcription 00:30:49.520 --> 00:30:52.250 of the genetic code. 00:30:52.250 --> 00:30:55.040 And then we have the macromolecular machine, 00:30:55.040 --> 00:31:02.950 the ribosome that allows for translation of this method 00:31:02.950 --> 00:31:04.925 message to give us a polypeptide chain. 00:31:10.070 --> 00:31:14.180 So some linear sequence of amino acids. 00:31:14.180 --> 00:31:16.550 And then what happens? 00:31:16.550 --> 00:31:18.770 We need to get from a polypeptide chain 00:31:18.770 --> 00:31:20.750 to some functional unit. 00:31:20.750 --> 00:31:26.390 And so there's a whole number of interesting players 00:31:26.390 --> 00:31:29.600 that are involved in protein folding. 00:31:29.600 --> 00:31:40.470 So we have folding, which is enabled by chaperones, 00:31:40.470 --> 00:31:44.560 is what we call these proteins that facilitate folding. 00:31:44.560 --> 00:31:50.230 And that's going to give us some structure that 00:31:50.230 --> 00:31:56.930 has some function here. 00:31:56.930 --> 00:32:00.920 And this protein has some lifetime in the cell. 00:32:00.920 --> 00:32:03.200 So at some time, for some reason, 00:32:03.200 --> 00:32:07.220 it will be time for this protein to get degraded. 00:32:07.220 --> 00:32:09.620 In which case, we need machinery that 00:32:09.620 --> 00:32:14.330 will facilitate the process to break down this folded protein 00:32:14.330 --> 00:32:16.190 into smaller fragments-- whether that 00:32:16.190 --> 00:32:19.700 be individual amino acids, or short polypeptide chains 00:32:19.700 --> 00:32:22.520 of seven to eight amino acids. 00:32:22.520 --> 00:32:33.990 So from here, we have degradation 00:32:33.990 --> 00:32:38.070 to give us small fragments. 00:32:38.070 --> 00:32:44.970 And the players here are proteases and chambers of doom, 00:32:44.970 --> 00:32:46.620 one of which is the proteasome. 00:32:51.290 --> 00:32:52.850 And actually, I forgot to mention 00:32:52.850 --> 00:32:56.690 there will be a second guest lecturer in recitation 00:32:56.690 --> 00:32:59.570 this year, Reuben Saunders, who is a senior, 00:32:59.570 --> 00:33:01.670 and does research in the Sauer Lab 00:33:01.670 --> 00:33:05.150 on one of these chambers of doom, called ClpXP. 00:33:05.150 --> 00:33:08.000 And so he'll present on single molecule methods, 00:33:08.000 --> 00:33:11.570 and fluorescence methods to study how this degradation 00:33:11.570 --> 00:33:12.310 chamber works. 00:33:12.310 --> 00:33:13.790 So that will be really exciting. 00:33:13.790 --> 00:33:16.950 He was a student in our course two years ago. 00:33:16.950 --> 00:33:18.750 So let's just take a look. 00:33:18.750 --> 00:33:20.480 We have the ribosome here. 00:33:23.290 --> 00:33:27.310 What are the structural features of this macromolecular machine, 00:33:27.310 --> 00:33:29.140 and how does it do its job? 00:33:29.140 --> 00:33:33.100 We'll look at a number of seminal studies that were done. 00:33:33.100 --> 00:33:37.300 And it is truly fascinating and incredible. 00:33:37.300 --> 00:33:39.670 What about protein folding? 00:33:39.670 --> 00:33:43.730 So look at this macromolecular machine here, 00:33:43.730 --> 00:33:47.650 GroEL, GroES, look at how big this is. 00:33:47.650 --> 00:33:56.320 So how does this chaperone allow some nascent polypeptide 00:33:56.320 --> 00:33:58.750 that's unfolded or partially folded 00:33:58.750 --> 00:34:01.510 to obtain its native structure? 00:34:01.510 --> 00:34:04.210 And there is many details in this depiction here 00:34:04.210 --> 00:34:06.160 that probably aren't apparent yet. 00:34:06.160 --> 00:34:12.610 But by the time we're done with module two, it will be there. 00:34:12.610 --> 00:34:14.929 Protein degradation. 00:34:14.929 --> 00:34:22.120 So here is just a cartoon-type depiction of a chamber of doom 00:34:22.120 --> 00:34:26.920 and its accessory protein from E. coli, ClpZ, ClpP. 00:34:26.920 --> 00:34:29.290 So look, we have a folded protein here, it's 00:34:29.290 --> 00:34:33.340 a beta barrel, our friend GFP that emits green light. 00:34:33.340 --> 00:34:37.719 And somehow, this protein gets threaded through ClpX, 00:34:37.719 --> 00:34:39.400 enters this chamber-- 00:34:39.400 --> 00:34:41.560 which has multiple protease active sites-- 00:34:41.560 --> 00:34:44.440 and that protein gets all degraded. 00:34:44.440 --> 00:34:46.159 So how does this work? 00:34:46.159 --> 00:34:50.620 How did ClpX and P work together to allow degradation 00:34:50.620 --> 00:34:53.920 of this condemned protein? 00:34:53.920 --> 00:34:56.350 And then finally, where I'll close is on something 00:34:56.350 --> 00:34:59.140 I think a little bit different for most everyone, 00:34:59.140 --> 00:35:02.590 and it's a type of template-driven polymerization 00:35:02.590 --> 00:35:05.170 involved in the synthesis of small molecules 00:35:05.170 --> 00:35:07.420 like penicillins and erythromycins. 00:35:07.420 --> 00:35:09.880 So these are antibiotics. 00:35:09.880 --> 00:35:13.420 So how do we get at molecules like these 00:35:13.420 --> 00:35:16.390 from simple amino acid precursors, 00:35:16.390 --> 00:35:20.860 or precursors like those you've seen in fatty acid biosynthesis 00:35:20.860 --> 00:35:21.640 here? 00:35:21.640 --> 00:35:25.343 And often, these are described as assembly lines. 00:35:25.343 --> 00:35:27.760 And something we'll just need to keep in mind in this unit 00:35:27.760 --> 00:35:31.090 is, are these proteins really acting like an assembly line, 00:35:31.090 --> 00:35:34.690 or is this just a way to help us think about the templates 00:35:34.690 --> 00:35:36.160 and what's going on here? 00:35:36.160 --> 00:35:38.290 So that's where we'll close. 00:35:38.290 --> 00:35:41.350 OK, so with that I'll finish up, and on Friday 00:35:41.350 --> 00:35:43.780 we'll begin with looking at the structure 00:35:43.780 --> 00:35:46.740 of the prokaryotic ribosome.