WEBVTT 00:00:00.500 --> 00:00:02.820 The following content is provided under a Creative 00:00:02.820 --> 00:00:04.360 Commons license. 00:00:04.360 --> 00:00:06.660 Your support will help MIT OpenCourseWare 00:00:06.660 --> 00:00:11.020 continue to offer high-quality educational resources for free. 00:00:11.020 --> 00:00:13.650 To make a donation or view additional materials 00:00:13.650 --> 00:00:17.600 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.600 --> 00:00:18.550 at ocw.mit.edu. 00:00:25.880 --> 00:00:29.630 ELIZABETH NOLAN: We're going to move on with GroEL/GroES 00:00:29.630 --> 00:00:33.860 and a few more comments about where we closed yesterday 00:00:33.860 --> 00:00:35.570 and then talk about experiments that 00:00:35.570 --> 00:00:37.730 were done to determine what polypeptides 00:00:37.730 --> 00:00:40.257 are folded by this machinery. 00:00:40.257 --> 00:00:41.090 So I'm just curious. 00:00:41.090 --> 00:00:44.390 Has anyone stuck trigger factor or GroEL into PubMed 00:00:44.390 --> 00:00:47.070 to see how many hits you get? 00:00:47.070 --> 00:00:51.260 Yeah, so Rebecca's question yesterday, or on Monday, 00:00:51.260 --> 00:00:54.110 about trigger factor and active versus passive folding 00:00:54.110 --> 00:00:56.247 motivated me to take a look. 00:00:56.247 --> 00:00:57.830 So just to give you some scope, if you 00:00:57.830 --> 00:01:00.110 put trigger factor in PubMed, as of last night, 00:01:00.110 --> 00:01:05.860 there's 11,810 hits there. 00:01:05.860 --> 00:01:09.650 GroEL is closer to 2,000 to 3,000-- in that range. 00:01:09.650 --> 00:01:12.890 If you put trigger factor active folding, 00:01:12.890 --> 00:01:15.200 you end up with 34 hits. 00:01:15.200 --> 00:01:17.360 Most of those are about using trigger factor 00:01:17.360 --> 00:01:19.410 in protein overexpression. 00:01:19.410 --> 00:01:23.130 So if you also express trigger factor, does that help? 00:01:23.130 --> 00:01:26.240 And it looked like there was one paper in those 34 00:01:26.240 --> 00:01:30.980 that suggests an active folding role for one of the domains. 00:01:30.980 --> 00:01:33.680 But that is just looking at an abstract. 00:01:33.680 --> 00:01:37.760 And so, the point there is there are many, many studies that 00:01:37.760 --> 00:01:41.340 consider these chaperones and a huge literature to search. 00:01:41.340 --> 00:01:44.420 So what we're able to cover here is really just the tip 00:01:44.420 --> 00:01:47.700 of the iceberg for that. 00:01:47.700 --> 00:01:51.140 There's also a new review out on GroEL/GroES, 00:01:51.140 --> 00:01:52.850 which is not required reading, but we're 00:01:52.850 --> 00:01:55.340 posting it on Stellar. 00:01:55.340 --> 00:01:57.770 So it just came out last month, and I really 00:01:57.770 --> 00:01:59.390 enjoyed reading this review. 00:01:59.390 --> 00:02:01.100 I thought they did a very good job 00:02:01.100 --> 00:02:06.530 of talking about current questions that are unanswered 00:02:06.530 --> 00:02:09.380 yet in terms of models and presenting different models 00:02:09.380 --> 00:02:12.560 for how this folding chamber works-- so passive 00:02:12.560 --> 00:02:14.760 versus active, for instance. 00:02:14.760 --> 00:02:18.620 And they also give a summary of the substrate scope-- 00:02:18.620 --> 00:02:21.650 so the experiment we'll talk about today. 00:02:21.650 --> 00:02:23.660 So where we left off last time, we 00:02:23.660 --> 00:02:27.320 went over the structure of this folding chamber 00:02:27.320 --> 00:02:30.570 and here's just another depiction of the overview. 00:02:30.570 --> 00:02:35.280 So effectively, we have to back-to-back heptamer rings 00:02:35.280 --> 00:02:37.700 as shown here. 00:02:37.700 --> 00:02:41.150 Some polypeptide in its non-native state can bind. 00:02:41.150 --> 00:02:44.840 It initially binds up at the top by these apical domains, 00:02:44.840 --> 00:02:47.250 and there are some hydrophobic interactions. 00:02:47.250 --> 00:02:51.250 OK, ATP also binds, and we have all seven ATPs 00:02:51.250 --> 00:02:55.970 found within one ring, the ring that has the polypeptide. 00:02:55.970 --> 00:02:59.480 OK, we see the lid come on, and then this polypeptide 00:02:59.480 --> 00:03:04.340 has some time, a residency time, in this chamber to fold. 00:03:04.340 --> 00:03:05.900 And then after the residency time, 00:03:05.900 --> 00:03:09.890 which is generally quoted on the order of 6 to 10 seconds, 00:03:09.890 --> 00:03:11.790 the lid comes off, and it gets ejected. 00:03:11.790 --> 00:03:14.420 And during that time, the ATPs are hydrolyzed. 00:03:14.420 --> 00:03:18.770 So somehow, this ATP hydrolysis gives conformational changes 00:03:18.770 --> 00:03:20.390 that drive this cycle. 00:03:20.390 --> 00:03:23.180 OK, and then we see, again, we flip 00:03:23.180 --> 00:03:26.300 to having function in the other ring. 00:03:26.300 --> 00:03:29.480 So one point to make involved cooperativity, 00:03:29.480 --> 00:03:32.420 so I hope you've all seen cooperativity before, probably 00:03:32.420 --> 00:03:34.970 in the context of hemoglobin. 00:03:34.970 --> 00:03:38.630 We have examples here of positive cooperativity 00:03:38.630 --> 00:03:40.940 and negative cooperativity. 00:03:40.940 --> 00:03:46.260 So within one heptamer ring, ATP binds to all seven subunits. 00:03:49.070 --> 00:03:51.230 So that's positive cooperativity. 00:03:51.230 --> 00:03:53.840 And then we can think about negative cooperativity 00:03:53.840 --> 00:03:55.700 between the two rings, where we only 00:03:55.700 --> 00:03:57.980 have ATPs bound to one ring. 00:03:57.980 --> 00:04:02.300 So the other heptamer ring will not have ATP bound here. 00:04:05.250 --> 00:04:09.650 So what is happening inside this chamber? 00:04:09.650 --> 00:04:11.990 The polypeptide enters the chamber, 00:04:11.990 --> 00:04:15.140 and it's given this protected environment to fold. 00:04:15.140 --> 00:04:17.990 And we saw that when the GroES lid comes in 00:04:17.990 --> 00:04:21.050 that the hydrophilic nature, hydrophobic nature 00:04:21.050 --> 00:04:22.310 of the interior changes. 00:04:22.310 --> 00:04:24.770 And it becomes more hydrophilic. 00:04:24.770 --> 00:04:28.130 So I just want to point out-- and this also builds upon 00:04:28.130 --> 00:04:30.500 Rebecca's question from last time-- 00:04:30.500 --> 00:04:32.510 is this passive folding in the chamber 00:04:32.510 --> 00:04:34.610 so effectively in Anfinsen's cage, 00:04:34.610 --> 00:04:38.870 where the primary sequence dictates the trajectory? 00:04:38.870 --> 00:04:42.140 Or does the actual chamber itself play a role? 00:04:42.140 --> 00:04:44.540 So that would be active folding. 00:04:44.540 --> 00:04:49.580 And effectively, is there forced unfolding or refolding 00:04:49.580 --> 00:04:51.440 by GroEL itself? 00:04:51.440 --> 00:04:53.960 So perhaps the apical domains can 00:04:53.960 --> 00:04:56.450 force unfolding before polypeptide 00:04:56.450 --> 00:04:57.890 is released into the chamber. 00:04:57.890 --> 00:05:01.340 And the cartoon that was just up indicated that to some degree. 00:05:01.340 --> 00:05:05.330 Maybe the cavity walls are involved. 00:05:05.330 --> 00:05:07.490 And what I would say is that the pendulum on this 00:05:07.490 --> 00:05:10.490 has swayed quite a bit over the years in terms of 00:05:10.490 --> 00:05:13.640 whether or not GroEL is a passive folding cage 00:05:13.640 --> 00:05:16.340 or actively involved in folding. 00:05:16.340 --> 00:05:21.680 And some of the debates in the literature 00:05:21.680 --> 00:05:25.430 have resulted from experimental set-up 00:05:25.430 --> 00:05:27.770 that may bias results to indicate 00:05:27.770 --> 00:05:28.853 one thing or the other. 00:05:28.853 --> 00:05:30.770 And that's something the community is striving 00:05:30.770 --> 00:05:32.930 to work out these days. 00:05:32.930 --> 00:05:35.480 And I'll talk about that a bit more on the next slide. 00:05:35.480 --> 00:05:39.710 But I'll just note-- these questions are still there, 00:05:39.710 --> 00:05:41.480 and the recent review I just noted 00:05:41.480 --> 00:05:44.960 discusses these questions. 00:05:44.960 --> 00:05:47.300 There was a study just a few years ago 00:05:47.300 --> 00:05:52.310 that was performed with very dilute polypeptide substrate-- 00:05:52.310 --> 00:05:54.500 so below one nanomolar. 00:05:54.500 --> 00:05:57.500 And what they conclude from this study 00:05:57.500 --> 00:06:01.820 is that GroEL is involved in active folding 00:06:01.820 --> 00:06:05.200 of a maltose-binding protein mutant. 00:06:05.200 --> 00:06:06.950 One question I'll just spring up with this 00:06:06.950 --> 00:06:10.790 is, maltose-binding protein is a nice model polypeptide, 00:06:10.790 --> 00:06:13.370 but what happens for a native GroEL substrate? 00:06:13.370 --> 00:06:16.310 And is there utility in studying those? 00:06:16.310 --> 00:06:20.810 So why have I emphasized this dilute protein sample point 00:06:20.810 --> 00:06:21.620 here? 00:06:21.620 --> 00:06:24.200 So what happened in some early work, 00:06:24.200 --> 00:06:26.930 in terms of studies that were done to try to differentiate 00:06:26.930 --> 00:06:29.960 active or passive folding, is that there 00:06:29.960 --> 00:06:33.780 were some complexities in in vitro studies. 00:06:33.780 --> 00:06:39.620 So, here, I just have a cartoon of folding in the chamber. 00:06:39.620 --> 00:06:43.340 And if we think about only one polypeptide within the GroEL 00:06:43.340 --> 00:06:46.190 chamber, it's folding in isolation. 00:06:46.190 --> 00:06:48.380 So there's no possibility for it to form 00:06:48.380 --> 00:06:52.360 an aggregate or a ligamer with other polypeptides. 00:06:52.360 --> 00:06:55.070 It's all alone here. 00:06:55.070 --> 00:06:58.100 So this folding in the chamber avoids the complications 00:06:58.100 --> 00:06:59.600 of the folding landscape we talked 00:06:59.600 --> 00:07:02.780 about in the introductory lecture to this module. 00:07:02.780 --> 00:07:07.060 So what happens in aqueous solution, right? 00:07:07.060 --> 00:07:10.400 There's the possibility that, depending on your conditions, 00:07:10.400 --> 00:07:13.460 maybe there's some sort of aggregate that forms. 00:07:13.460 --> 00:07:15.350 And if this aggregate forms, what does that 00:07:15.350 --> 00:07:18.170 mean in terms of what you see? 00:07:18.170 --> 00:07:21.650 And so, in earlier work, there were 00:07:21.650 --> 00:07:24.170 some in vitro kinetic studies that 00:07:24.170 --> 00:07:27.860 indicated GroEL accelerates folding relative to folding 00:07:27.860 --> 00:07:30.530 in dilute aqueous solution. 00:07:30.530 --> 00:07:33.110 But some of these comparisons weren't appropriate, 00:07:33.110 --> 00:07:38.420 because as it turns out, oligomerization might compete 00:07:38.420 --> 00:07:40.280 with what you're watching for. 00:07:40.280 --> 00:07:43.760 And so, if there's some oligomerization happening, 00:07:43.760 --> 00:07:46.910 it might indicate that the rate is slower than you think. 00:07:46.910 --> 00:07:48.590 So there's ways to monitor for this. 00:07:48.590 --> 00:07:52.040 And it's just a point in terms of what control studies do 00:07:52.040 --> 00:07:55.220 you need to do to make sure your experimental setup is 00:07:55.220 --> 00:07:57.585 appropriate there. 00:07:57.585 --> 00:07:59.210 I think it'll be exciting to see what's 00:07:59.210 --> 00:08:02.540 to come in future years about this question 00:08:02.540 --> 00:08:05.390 and what kinds of biophysical techniques 00:08:05.390 --> 00:08:10.610 are applied, including single-molecule studies here. 00:08:10.610 --> 00:08:13.340 So where we're going to go, moving on, 00:08:13.340 --> 00:08:17.810 is to think about what actually are the substrates for GroEL. 00:08:17.810 --> 00:08:21.200 So what polypeptides get folded in this chamber? 00:08:21.200 --> 00:08:24.170 And how do we begin to address that question 00:08:24.170 --> 00:08:27.410 from the standpoint of what's happening in the cell? 00:08:27.410 --> 00:08:31.220 OK, so first, we're just going to consider some observations. 00:08:31.220 --> 00:08:34.580 And then we're going to go into the experiments here. 00:08:34.580 --> 00:08:37.610 So here are some observations. 00:08:37.610 --> 00:08:40.010 So the first one is that polypeptides, 00:08:40.010 --> 00:08:44.150 up to 60 kilodaltons, can fold in this chamber. 00:08:44.150 --> 00:08:45.620 So that's quite big-- 00:08:45.620 --> 00:08:48.560 60 kilodaltons. 00:08:48.560 --> 00:08:51.380 Some proteins or polypeptides need 00:08:51.380 --> 00:08:55.550 to enter the GroEL multiple times to be folded. 00:08:55.550 --> 00:08:59.270 So that means the chaperone has the ability to bind and release 00:08:59.270 --> 00:09:03.470 and re-bind the polypeptide here. 00:09:03.470 --> 00:09:07.370 So when studies are done in vitro, what's found 00:09:07.370 --> 00:09:11.750 is that almost all polypeptides interact with GroEL. 00:09:11.750 --> 00:09:15.320 So you just saw even an example of that 00:09:15.320 --> 00:09:18.020 in terms of this non-native maltose-binding protein. 00:09:18.020 --> 00:09:20.720 So many polypeptides will interact. 00:09:20.720 --> 00:09:24.140 And this really contrasts what's observed in the cell, 00:09:24.140 --> 00:09:30.455 where, in vivo GroEL is involved in only folding about 10% of E. 00:09:30.455 --> 00:09:32.510 coli proteins here. 00:09:32.510 --> 00:09:36.650 OK, so what observations three and four suggest 00:09:36.650 --> 00:09:39.590 is that GroEL has some preference 00:09:39.590 --> 00:09:42.680 for particular endogenous polypeptides. 00:09:42.680 --> 00:09:46.640 And what we want to answer is, what are these polypeptides, 00:09:46.640 --> 00:09:49.160 and what are their properties here? 00:09:49.160 --> 00:09:52.670 OK, so Hartl's group did some nice studies 00:09:52.670 --> 00:09:56.138 to look at this, what needs to be done. 00:09:56.138 --> 00:09:57.680 First of all, there needs to be a way 00:09:57.680 --> 00:10:00.710 to isolate the polypeptides that are interacting 00:10:00.710 --> 00:10:03.260 with GroEL in the cell. 00:10:03.260 --> 00:10:06.290 And then, once these polypeptides are isolated, 00:10:06.290 --> 00:10:08.450 they need to be analyzed in order 00:10:08.450 --> 00:10:11.640 to learn about their identity and properties. 00:10:11.640 --> 00:10:14.120 OK, so we're going to look at experiments 00:10:14.120 --> 00:10:17.180 that were done to address this. 00:10:17.180 --> 00:10:20.120 And they involve pulse-chase labeling 00:10:20.120 --> 00:10:24.290 of newly synthesized proteins, amino precipitation, 00:10:24.290 --> 00:10:25.860 and analysis here. 00:10:25.860 --> 00:10:30.502 So in terms of addressing what are these substrates, 00:10:30.502 --> 00:10:32.460 we're going to begin with pulse-chase labeling. 00:10:42.980 --> 00:10:47.885 OK, so basically, the goal of this experiment 00:10:47.885 --> 00:10:49.260 and why we're starting here is we 00:10:49.260 --> 00:11:06.740 want to determine which proteins interact with GroEL. 00:11:06.740 --> 00:11:08.870 And, in addition to which proteins, 00:11:08.870 --> 00:11:10.850 we want to determine how long they interact. 00:11:27.946 --> 00:11:31.320 OK, so what is the experiment? 00:11:31.320 --> 00:11:32.880 These experiments are going to be 00:11:32.880 --> 00:11:36.080 done with like E. coli cells. 00:11:36.080 --> 00:11:38.220 So we want to know what's happening in the cell. 00:11:38.220 --> 00:11:39.750 So imagine we have an E. coli. 00:11:43.250 --> 00:11:47.490 And so these bacteria are grown in some culture medium. 00:11:47.490 --> 00:11:49.110 And the trick here is that they're 00:11:49.110 --> 00:11:55.140 going to be grown in medium that's depleted in methionine. 00:11:55.140 --> 00:12:11.350 So incubate, or grow, in medium with no methionine. 00:12:11.350 --> 00:12:16.850 OK, so effectively, we're depleting them 00:12:16.850 --> 00:12:19.610 of that amino acid. 00:12:19.610 --> 00:12:23.320 OK, so then after some period of growth, 00:12:23.320 --> 00:12:25.040 what are we going to do? 00:12:25.040 --> 00:12:29.440 We're going to spike the culture with radiolabeled methionine. 00:12:29.440 --> 00:12:31.720 And this is the pulse. 00:12:31.720 --> 00:12:35.860 So we're going to add 35S methionine. 00:12:38.950 --> 00:12:42.600 And we're then going to incubate for 15 seconds. 00:12:46.650 --> 00:12:51.330 OK, and so that's the pulse with a radiolabeled amino acid. 00:12:51.330 --> 00:12:52.650 Then what are we going to do? 00:12:52.650 --> 00:12:56.550 And after we go through the steps, we'll go through why. 00:12:56.550 --> 00:12:58.980 After this stage, we're going to add 00:12:58.980 --> 00:13:01.300 excess unlabeled methionine. 00:13:13.980 --> 00:13:19.900 And we're going to then continue this culture for 10 minutes. 00:13:19.900 --> 00:13:24.690 OK, this is the chase here. 00:13:24.690 --> 00:13:28.290 And during this chase period, basically, samples 00:13:28.290 --> 00:13:35.765 will be taken at varying time points. 00:13:40.060 --> 00:13:42.910 OK, and then, at some point, we're just going to stop this. 00:13:42.910 --> 00:13:50.760 OK, so just say, stop culture and experiment. 00:13:56.270 --> 00:14:00.530 So what's happening in each of these steps? 00:14:00.530 --> 00:14:02.990 And why are we doing this? 00:14:02.990 --> 00:14:09.530 So what we want to do is think about newly translated 00:14:09.530 --> 00:14:11.030 polypeptides. 00:14:11.030 --> 00:14:13.730 OK, so we have a living E. coli. 00:14:13.730 --> 00:14:14.750 It has ribosomes. 00:14:14.750 --> 00:14:17.990 And these ribosomes are going to be synthesizing polypeptides 00:14:17.990 --> 00:14:21.210 over the course of this experiment. 00:14:21.210 --> 00:14:35.850 So during the pulse period, all proteins, or all polypeptides, 00:14:35.850 --> 00:14:42.090 synthesized are radiolabeled. 00:14:51.210 --> 00:14:53.840 Right, because the methionine has been depleted 00:14:53.840 --> 00:14:55.580 from the culture medium. 00:14:55.580 --> 00:14:57.920 And so effectively, the methionine 00:14:57.920 --> 00:15:03.900 that these organisms are seeing are the S35-labeled methionine. 00:15:03.900 --> 00:15:07.970 And all polypeptides have an informal methionine 00:15:07.970 --> 00:15:11.120 from the initiator tRNA and what other methionines 00:15:11.120 --> 00:15:13.310 are in the sequence. 00:15:13.310 --> 00:15:17.330 So, if we think about doing this for 15 seconds, 00:15:17.330 --> 00:15:21.130 and we think about the translation rate, which 00:15:21.130 --> 00:15:24.260 I gave as 6 to 20 amino acids per second 00:15:24.260 --> 00:15:26.690 when we were discussing the ribosome, 00:15:26.690 --> 00:15:30.710 we want to think about how long are these polypeptides going 00:15:30.710 --> 00:15:31.370 to be? 00:15:31.370 --> 00:15:39.530 So we have a translation rate of 6 to 20 amino acids per second. 00:15:39.530 --> 00:15:45.260 OK, so, if we think about 15 seconds of a pulse, 00:15:45.260 --> 00:15:48.320 we're getting polypeptides on the order 00:15:48.320 --> 00:15:56.800 of 90 to 300 amino acids synthesized during that time. 00:16:00.850 --> 00:16:05.080 So newly synthesized polypeptides in these 15 00:16:05.080 --> 00:16:07.030 seconds are radiolabeled. 00:16:07.030 --> 00:16:09.190 What happens next? 00:16:09.190 --> 00:16:12.730 OK, we have this chase period where we flood the system 00:16:12.730 --> 00:16:14.860 with unlabeled methionine here. 00:16:24.160 --> 00:16:27.640 Why are we doing this? 00:16:27.640 --> 00:16:30.280 So certainly, there are some polypeptides 00:16:30.280 --> 00:16:35.320 that are longer than 300 amino acids that still need time 00:16:35.320 --> 00:16:37.120 to be synthesized. 00:16:37.120 --> 00:16:41.260 And if there's new peptides being synthesized 00:16:41.260 --> 00:16:43.690 that start in this stage, we won't see them, 00:16:43.690 --> 00:16:45.470 because this unlabeled methionine 00:16:45.470 --> 00:16:49.210 is in vast access over the radiolabeled methionine that 00:16:49.210 --> 00:16:52.060 was added early. 00:16:52.060 --> 00:17:07.920 So here, we have, the synthesis of larger polypeptides 00:17:07.920 --> 00:17:08.970 can be completed. 00:17:20.900 --> 00:17:35.840 And we have, no longer producing radiolabeled new peptides. 00:17:41.910 --> 00:17:44.880 OK, so this allows us to only see 00:17:44.880 --> 00:17:47.550 the peptides that were radiolabeled during this pulse 00:17:47.550 --> 00:17:48.690 period here. 00:17:55.160 --> 00:17:57.230 So what are we going to do in terms 00:17:57.230 --> 00:18:00.710 of the sampling at various time points? 00:18:08.900 --> 00:18:12.800 So let's say we want to sample at one minute, five minutes, 00:18:12.800 --> 00:18:13.490 ten minutes. 00:18:13.490 --> 00:18:15.330 What do we need to do? 00:18:15.330 --> 00:18:18.020 So can we just aliquot some of these E. coli 00:18:18.020 --> 00:18:19.895 and put them on our bench? 00:18:32.570 --> 00:18:37.050 We could, but that's not going to be very helpful to us, 00:18:37.050 --> 00:18:40.910 because what we want to do is stop the translation 00:18:40.910 --> 00:18:45.325 machinery and all of the cellular machinery here. 00:18:45.325 --> 00:18:46.950 AUDIENCE: You need some kind of clench? 00:18:46.950 --> 00:18:48.980 ELIZABETH NOLAN: Yeah, we need a clench. 00:18:48.980 --> 00:18:50.810 And not only do we need a clench, 00:18:50.810 --> 00:18:53.600 we're dealing with a living organism too, right? 00:18:53.600 --> 00:18:57.980 So we need to break open the E. coli in whatever this condition 00:18:57.980 --> 00:19:00.320 is to stop the reaction. 00:19:00.320 --> 00:19:10.208 OK, so we're going to take aliquots 00:19:10.208 --> 00:19:11.700 at varying time points. 00:19:16.150 --> 00:19:18.940 And basically, we care about time, 00:19:18.940 --> 00:19:22.120 so you have to immediately lyse, or break open, the cells. 00:19:27.190 --> 00:19:29.575 And this was done in the presence of EDTA. 00:19:39.040 --> 00:19:39.978 So what is EDTA? 00:19:39.978 --> 00:19:41.478 AUDIENCE: Ethylenediaminetetraacetic 00:19:41.478 --> 00:19:41.978 acid. 00:19:41.978 --> 00:19:44.570 ELIZABETH NOLAN: Yeah, ethylenediaminetetraacetic 00:19:44.570 --> 00:19:45.070 acid. 00:19:45.070 --> 00:19:47.890 So it's the chelator. 00:19:47.890 --> 00:19:52.300 And why might this lysis be done in the presence of this metal 00:19:52.300 --> 00:19:54.604 chelator? 00:19:54.604 --> 00:19:59.810 AUDIENCE: [INAUDIBLE] processes like-- 00:19:59.810 --> 00:20:05.330 [INAUDIBLE] magnesium, which would help [INAUDIBLE] 00:20:05.330 --> 00:20:10.520 AUDIENCE: Are the proteases that are not binding? 00:20:10.520 --> 00:20:12.890 ELIZABETH NOLAN: There certainly are zinc proteases. 00:20:12.890 --> 00:20:16.610 So that that's one class of protease. 00:20:16.610 --> 00:20:21.890 So EDTA will chelate many, many different metals. 00:20:21.890 --> 00:20:26.180 The main point here is we want to stop stop translation, 00:20:26.180 --> 00:20:29.470 shut down processes here. 00:20:29.470 --> 00:20:32.665 OK, so we have these samples. 00:20:35.470 --> 00:20:36.685 What do we need to do next? 00:20:47.560 --> 00:21:06.370 We need to detect these newly synthesized proteins that 00:21:06.370 --> 00:21:14.200 interact with GroEL. 00:21:14.200 --> 00:21:17.430 And we want to do this at each time point. 00:21:17.430 --> 00:21:19.200 So how are we going to do this? 00:21:19.200 --> 00:21:22.550 We have a very complex mixture that has 00:21:22.550 --> 00:21:24.440 all of the cellular components. 00:21:36.210 --> 00:21:41.330 So the next step in this will be immunoprecipitation. 00:21:54.850 --> 00:21:58.270 And so, what will happen in immunoprecipitation 00:21:58.270 --> 00:22:02.470 in these experiments is that the researchers had an antibody 00:22:02.470 --> 00:22:04.210 that binds to GroEL. 00:22:04.210 --> 00:22:07.600 And this antibody was put on a bead 00:22:07.600 --> 00:22:12.040 and used to fish out GroEL from this complex mixture. 00:22:12.040 --> 00:22:15.190 And we need to talk about these antibodies a little more. 00:22:15.190 --> 00:22:20.560 But just in starting, I imagine there's a bead. 00:22:20.560 --> 00:22:26.870 And we think about antibodies as being Y-shaped biomolecules. 00:22:26.870 --> 00:22:28.030 So here, we have a GroEL. 00:22:37.550 --> 00:22:41.240 And imagine that, in this mixture, 00:22:41.240 --> 00:22:46.430 we have GroEL that has some polypeptide bound. 00:22:52.810 --> 00:22:56.260 That's one of its endogenous substrates. 00:22:56.260 --> 00:23:05.420 So, if these are mixed together, then the antibody 00:23:05.420 --> 00:23:12.765 binds GroEL with the polypeptide attached. 00:23:16.620 --> 00:23:24.760 OK, here, we can imagine "capture" of this species 00:23:24.760 --> 00:23:29.500 here and using the bead to separate, say, 00:23:29.500 --> 00:23:32.230 by centrifugation. 00:23:32.230 --> 00:23:36.780 So let's think about this a little bit 00:23:36.780 --> 00:23:40.750 and a little background to have everyone up to speed. 00:23:40.750 --> 00:23:43.240 If you need to learn more about antibodies, 00:23:43.240 --> 00:23:47.710 please see a basic biology textbook for further details. 00:23:47.710 --> 00:23:49.870 But these are Y-shaped molecules that 00:23:49.870 --> 00:23:53.710 are produced by a type of immune cell called B cells. 00:23:53.710 --> 00:23:55.870 And they're used by the immune system 00:23:55.870 --> 00:24:01.270 to detect foreign biomolecules and help to neutralize them. 00:24:01.270 --> 00:24:03.550 And so, in these, the tip of the Y 00:24:03.550 --> 00:24:07.990 contains the paratope that ideally binds specifically 00:24:07.990 --> 00:24:12.850 to a particular epitope-- in this case, GroEL here. 00:24:12.850 --> 00:24:15.880 And so, we often think about a lock-and-key model 00:24:15.880 --> 00:24:19.570 with antibody and think about the antibody binding its target 00:24:19.570 --> 00:24:24.140 with precision here. 00:24:24.140 --> 00:24:27.250 So for these experiments that were done, 00:24:27.250 --> 00:24:29.410 just realize the researchers had to come up 00:24:29.410 --> 00:24:31.510 with an antibody to GroEL. 00:24:31.510 --> 00:24:33.040 How is that done? 00:24:33.040 --> 00:24:36.390 They may have immunized, say, a rabbit 00:24:36.390 --> 00:24:39.910 or given a rabbit GroEL and allowed that rabbit 00:24:39.910 --> 00:24:40.990 to produce antibodies. 00:24:40.990 --> 00:24:44.110 And then they isolate the antibodies here. 00:24:44.110 --> 00:24:47.650 So something we want you to take home from this course 00:24:47.650 --> 00:24:51.890 is, yes, the antibodies should bind the target with precision. 00:24:51.890 --> 00:24:54.790 But there's huge problems in terms of use 00:24:54.790 --> 00:24:57.040 of antibodies in research. 00:24:57.040 --> 00:24:59.800 This is just the start of an article that was published 00:24:59.800 --> 00:25:01.270 last year around this time. 00:25:01.270 --> 00:25:03.670 And it's focused on pharma and clinical trials. 00:25:03.670 --> 00:25:05.890 But this is much more broad. 00:25:05.890 --> 00:25:09.850 And often, antibodies aren't as specific as indicated 00:25:09.850 --> 00:25:15.130 by the label on the container from the supplier here. 00:25:15.130 --> 00:25:16.960 And it's pretty dismal what they quote 00:25:16.960 --> 00:25:22.810 in this terms of how difficult it is to reproduce data here. 00:25:22.810 --> 00:25:24.910 So if you're going to use an antibody, 00:25:24.910 --> 00:25:30.100 you always need to test it to see whether it is selective 00:25:30.100 --> 00:25:33.040 or not for the species of interest 00:25:33.040 --> 00:25:35.740 that you want to detect there and have 00:25:35.740 --> 00:25:39.490 that information on hand so you don't misinterpret your data 00:25:39.490 --> 00:25:43.060 here for that. 00:25:43.060 --> 00:25:48.430 So what are the steps for this immunoprecipitation? 00:25:48.430 --> 00:25:51.910 Basically, as shown on the board, 00:25:51.910 --> 00:25:54.670 beads will be functionalized with the antibody 00:25:54.670 --> 00:25:58.330 and then just added to the cell lysate. 00:25:58.330 --> 00:26:03.250 And the antibody can recognize GroEL. 00:26:03.250 --> 00:26:06.970 And the goal and hope are that whatever 00:26:06.970 --> 00:26:11.590 polypeptides are associated with GroEL are pulled down together. 00:26:11.590 --> 00:26:13.090 So that's something a bit incredible 00:26:13.090 --> 00:26:17.260 here that these polypeptides remain bound to GroEL 00:26:17.260 --> 00:26:19.900 during the steps of this process. 00:26:19.900 --> 00:26:22.360 You can imagine, if there's a low-affinity binder, 00:26:22.360 --> 00:26:23.890 it could be lost. 00:26:23.890 --> 00:26:26.200 So the sample can be centrifuged. 00:26:26.200 --> 00:26:28.810 And then, you can isolate these beads here. 00:26:31.450 --> 00:26:34.780 So, in cartoon form, a complex cell 00:26:34.780 --> 00:26:37.680 lysate in your microcentrifuge tube. 00:26:37.680 --> 00:26:41.710 You can add the antibody, centrifuge. 00:26:41.710 --> 00:26:44.170 And see, down here, we've pelleted the beads 00:26:44.170 --> 00:26:45.820 with GroEL attached. 00:26:45.820 --> 00:26:47.440 And then some sort of workup needs 00:26:47.440 --> 00:26:51.640 to be done to dissociate the protein, or polypeptide, 00:26:51.640 --> 00:26:52.960 substrates here. 00:26:52.960 --> 00:26:55.432 And then they can be analyzed. 00:26:55.432 --> 00:26:58.258 AUDIENCE: How long do they do that for? 00:26:58.258 --> 00:26:59.680 Do you know how many-- 00:26:59.680 --> 00:27:01.722 ELIZABETH NOLAN: How long do they centrifuge for? 00:27:01.722 --> 00:27:04.631 AUDIENCE: No, for the immunoprecipitation. 00:27:04.631 --> 00:27:05.570 Is it 30 minutes? 00:27:05.570 --> 00:27:06.070 Is it-- 00:27:06.070 --> 00:27:07.870 ELIZABETH NOLAN: I don't know how long the incubation 00:27:07.870 --> 00:27:08.370 time is. 00:27:08.370 --> 00:27:09.940 Need to go back to the experimental, 00:27:09.940 --> 00:27:13.180 but that's getting right back to this question as to how 00:27:13.180 --> 00:27:14.210 do they stay bound. 00:27:14.210 --> 00:27:15.708 AUDIENCE: How do they stay bound? 00:27:15.708 --> 00:27:16.625 ELIZABETH NOLAN: Yeah. 00:27:20.530 --> 00:27:21.490 So, see the point here. 00:27:21.490 --> 00:27:24.310 If you have a high-affinity complex, that's one thing. 00:27:24.310 --> 00:27:26.840 If you have low-affinity association between GroEL 00:27:26.840 --> 00:27:29.080 and the polypeptide, you can imagine 00:27:29.080 --> 00:27:32.360 it might get lost during this workup. 00:27:32.360 --> 00:27:37.240 And how much do we know about those affinities there? 00:27:37.240 --> 00:27:40.400 AUDIENCE: You said that they would just give rabbits GroEL, 00:27:40.400 --> 00:27:44.500 and hopefully antibodies would just happen. 00:27:44.500 --> 00:27:47.810 But if a rabbit's immune system encountered GroEL, 00:27:47.810 --> 00:27:50.884 would it actually see it as an antigen 00:27:50.884 --> 00:27:53.630 that it had to develop antibodies against? 00:27:53.630 --> 00:27:55.030 ELIZABETH NOLAN: So, yeah. 00:27:55.030 --> 00:27:56.500 So here's the point-- 00:27:56.500 --> 00:27:58.150 would it? 00:27:58.150 --> 00:28:03.880 So, if it's E. coli GroEL, would the rabbit recognize this, 00:28:03.880 --> 00:28:04.660 yes or no? 00:28:04.660 --> 00:28:08.200 And if no, then what can you do to provoke an antibody 00:28:08.200 --> 00:28:08.910 response? 00:28:08.910 --> 00:28:10.630 And so, what can be done is, say, 00:28:10.630 --> 00:28:13.990 you could take a GroEL subunit and attach that 00:28:13.990 --> 00:28:15.970 to something immunogenic. 00:28:15.970 --> 00:28:18.520 So there are carrier proteins that 00:28:18.520 --> 00:28:21.100 will mount an immune response. 00:28:21.100 --> 00:28:25.120 So one of the subunits of cholera toxin 00:28:25.120 --> 00:28:27.490 is an example that can be used. 00:28:27.490 --> 00:28:29.920 And then the idea is you're mounting an immune response 00:28:29.920 --> 00:28:31.270 against that carrier protein. 00:28:31.270 --> 00:28:35.170 But you'll also get antibodies to whatever is attached. 00:28:35.170 --> 00:28:37.480 So that's another strategy for doing it 00:28:37.480 --> 00:28:39.370 if direct injection doesn't work. 00:28:39.370 --> 00:28:42.790 And too, not going off on a big tangent, 00:28:42.790 --> 00:28:45.290 but there are some decisions that need to be made. 00:28:45.290 --> 00:28:47.920 So would they use the full-length GroEL? 00:28:47.920 --> 00:28:51.160 Or maybe they would just use a polypeptide region, 00:28:51.160 --> 00:28:55.060 like some shorter polypeptide that's a portion of GroEL. 00:28:55.060 --> 00:28:56.710 So there's a lot of possibilities 00:28:56.710 --> 00:28:58.960 there in terms of what you use to generate 00:28:58.960 --> 00:29:06.190 the antibody for that there. 00:29:06.190 --> 00:29:10.930 And it's something that a lot of companies do these days. 00:29:10.930 --> 00:29:15.490 You can send them your protein or your polypeptide fragment. 00:29:15.490 --> 00:29:19.150 And they'll conjugate it to one of these carriers 00:29:19.150 --> 00:29:23.590 and treat the rabbits or whatever animal 00:29:23.590 --> 00:29:25.540 and then isolate those antibodies. 00:29:25.540 --> 00:29:29.770 And then they need to be characterized there for that. 00:29:29.770 --> 00:29:36.910 OK, so how are these samples going to be analyzed? 00:29:36.910 --> 00:29:38.710 That's the next step. 00:29:41.320 --> 00:29:45.810 So, for the analysis, effectively, we're 00:29:45.810 --> 00:29:48.010 going to have some mixture. 00:29:48.010 --> 00:29:50.440 And, at the onset, we don't really 00:29:50.440 --> 00:29:54.340 know how complicated this mixture will be. 00:29:54.340 --> 00:29:59.440 I told you initially that about 10% of E. coli polypeptides 00:29:59.440 --> 00:30:02.470 are thought to be substrates for GroEL, which 00:30:02.470 --> 00:30:04.990 is quite a large number if we think about the total number 00:30:04.990 --> 00:30:07.840 of proteins in E. coli. 00:30:07.840 --> 00:30:11.020 And the other point is we have this radiolabel, which 00:30:11.020 --> 00:30:14.110 we're going to use for detection there. 00:30:14.110 --> 00:30:16.750 OK, so, for analysis-- 00:30:35.530 --> 00:30:36.590 OK, there's two things. 00:30:36.590 --> 00:30:40.100 We need to separate these various polypeptides 00:30:40.100 --> 00:30:42.230 in each sample. 00:30:42.230 --> 00:30:45.770 And then we need to determine what their identities are here. 00:30:48.480 --> 00:31:16.855 So-- that were bound to GroEL from one another. 00:31:23.450 --> 00:31:28.400 OK, and then, we need to determine identities. 00:31:32.480 --> 00:31:34.070 And once we know the identities, we 00:31:34.070 --> 00:31:36.590 can think about their properties. 00:31:36.590 --> 00:31:39.770 And this needs to be done in every sample that was collected 00:31:39.770 --> 00:31:43.460 along this time course, which is also going to give 00:31:43.460 --> 00:31:45.870 some temporal information. 00:31:45.870 --> 00:31:48.870 So what are the methods that have been used? 00:31:48.870 --> 00:31:51.770 So, in order to separate the proteins in this complex 00:31:51.770 --> 00:31:56.090 sample, the method is a 2-D gel-- 00:31:56.090 --> 00:31:58.730 so 2-D gel electrophoresis. 00:32:06.500 --> 00:32:11.910 OK, and in terms of determining the identities, what's 00:32:11.910 --> 00:32:14.890 done, once these polypeptides are separated, 00:32:14.890 --> 00:32:23.260 is to do a protease digest and then mass spectrometry. 00:32:31.950 --> 00:32:40.540 Has anyone here ever run a 2-D gel or seen the equipment? 00:32:40.540 --> 00:32:41.860 One person. 00:32:41.860 --> 00:32:44.816 Has anyone heard of 2-D gels? 00:32:44.816 --> 00:32:45.940 Fair number. 00:32:45.940 --> 00:32:50.950 OK, so, we'll go over this briefly in terms of 2-D gel. 00:33:11.060 --> 00:33:23.090 So, in terms of 2-D gel electrophoresis, 00:33:23.090 --> 00:33:27.140 we talk about running these gels in two dimensions. 00:33:27.140 --> 00:33:29.540 And, in each dimension, we separate 00:33:29.540 --> 00:33:31.490 based on a different property. 00:33:31.490 --> 00:33:39.720 So, in the first dimension, the separation is based on charge. 00:33:52.400 --> 00:33:59.670 And effectively, we can talk about the pI of a protein. 00:33:59.670 --> 00:34:04.240 So the pI is the isoelectric point. 00:34:09.820 --> 00:34:13.960 And it's the pH where the net charge on the protein is zero. 00:34:27.500 --> 00:34:31.550 And so, the type of gel we use here 00:34:31.550 --> 00:34:35.869 is called isoelectric focusing, or IEF. 00:34:48.920 --> 00:34:54.920 And effectively, what's done is that the gel electrophoresis is 00:34:54.920 --> 00:34:57.410 done through a continuous and stable pH gradient. 00:35:27.190 --> 00:35:31.720 And, in this gel, the protein will migrate to a position 00:35:31.720 --> 00:35:33.560 where the pH corresponds to the pI. 00:35:37.140 --> 00:35:39.955 Then the anode is low pH and the cathode high pH. 00:35:43.780 --> 00:35:48.130 So that's quite different than SDS, where, in an SDS-PAGE gel, 00:35:48.130 --> 00:35:53.230 we're coating the protein with negative charge. 00:35:53.230 --> 00:36:00.340 So then, the second dimension is something most of us 00:36:00.340 --> 00:36:02.040 are familiar with, is SDS-PAGE. 00:36:08.810 --> 00:36:14.150 And so, what happens in SDS-PAGE? 00:36:14.150 --> 00:36:24.490 We have separation based on size here-- 00:36:27.850 --> 00:36:29.140 on molecular weight. 00:36:34.280 --> 00:36:38.270 So has anyone not run an SDS-PAGE gel? 00:36:38.270 --> 00:36:39.560 And this is totally fine. 00:36:39.560 --> 00:36:41.200 I never ran one till I was a postdoc. 00:36:41.200 --> 00:36:45.390 So it's not something to be ashamed about if you haven't. 00:36:45.390 --> 00:36:46.750 OK, so everyone has. 00:36:46.750 --> 00:36:51.100 So what's the ratio of SDS molecules to amino acids? 00:36:54.110 --> 00:36:55.960 So if you take your protein sample 00:36:55.960 --> 00:37:02.582 and you put it in your loading buffer and run your SDS-PAGE, 00:37:02.582 --> 00:37:03.790 what is the ratio of binding? 00:37:14.650 --> 00:37:16.642 What is SDS? 00:37:16.642 --> 00:37:20.050 AUDIENCE: Sodium dodecyl sulfate. 00:37:20.050 --> 00:37:21.670 ELIZABETH NOLAN: And what does it do? 00:37:21.670 --> 00:37:23.990 What happens to your protein in SDS? 00:37:23.990 --> 00:37:25.250 AUDIENCE: Denatures it. 00:37:25.250 --> 00:37:26.542 ELIZABETH NOLAN: OK, what else? 00:37:26.542 --> 00:37:27.430 So it's a denaturant. 00:37:27.430 --> 00:37:28.597 So it denatures the protein. 00:37:33.120 --> 00:37:35.070 So why does SDS-PAGE let you separate 00:37:35.070 --> 00:37:37.210 based on molecular weight, more or less? 00:37:37.210 --> 00:37:39.720 AUDIENCE: It coats the protein, more or less, 00:37:39.720 --> 00:37:41.983 uniformly with negative charge. 00:37:41.983 --> 00:37:42.900 ELIZABETH NOLAN: Yeah. 00:37:42.900 --> 00:37:45.312 AUDIENCE: Do we know the exact ratio of binding? 00:37:45.312 --> 00:37:47.520 ELIZABETH NOLAN: Yeah, so what's the ratio of binding 00:37:47.520 --> 00:37:50.370 that can be done in terms of grams 00:37:50.370 --> 00:37:53.880 of SDS per grams of protein or number of SDS 00:37:53.880 --> 00:37:56.490 molecules per amino acid. 00:37:56.490 --> 00:37:59.330 What is it? 00:37:59.330 --> 00:38:01.832 And there'll be some error, but there's approximates. 00:38:01.832 --> 00:38:03.540 But it's something to think about, right? 00:38:03.540 --> 00:38:06.120 You're putting your sample into this. 00:38:06.120 --> 00:38:11.010 So it's about 1.4 grams of SDS per gram of protein. 00:38:11.010 --> 00:38:13.470 That's the ratio there. 00:38:13.470 --> 00:38:18.360 And as said, the idea is that SDS is giving the protein 00:38:18.360 --> 00:38:20.520 a large net negative charge. 00:38:20.520 --> 00:38:22.980 So it's going to override whatever the intrinsic charge 00:38:22.980 --> 00:38:25.410 is of the protein. 00:38:25.410 --> 00:38:30.270 And so, it gives all proteins a similar mass-to-charge ratio 00:38:30.270 --> 00:38:31.500 here. 00:38:31.500 --> 00:38:33.000 With that said, sometimes, there are 00:38:33.000 --> 00:38:35.790 proteins that migrate in the gel in a manner that's 00:38:35.790 --> 00:38:38.040 not reflective of their molecular weight. 00:38:38.040 --> 00:38:40.390 That's just something to keep an eye out on. 00:38:40.390 --> 00:38:42.810 So within the slides that will be posted on Stellar, 00:38:42.810 --> 00:38:44.790 there'll be some background information 00:38:44.790 --> 00:38:46.490 about both of these methods-- 00:38:46.490 --> 00:38:49.380 the IEF gel and SDS-PAGE, which I 00:38:49.380 --> 00:38:53.330 encourage you to take a look there. 00:38:53.330 --> 00:38:57.570 OK, so back to the 2-D gel-- 00:38:57.570 --> 00:38:59.430 how is this actually going to be run? 00:39:08.660 --> 00:39:10.640 So it's one gel. 00:39:10.640 --> 00:39:16.760 First, it needs to run the IEF gel. 00:39:16.760 --> 00:39:18.530 And you need a special apparatus for. 00:39:18.530 --> 00:39:21.080 This it's called a cylinder, or tube, gel-- 00:39:21.080 --> 00:39:23.780 so not flat like what you're all accustomed to for SDS-PAGE. 00:39:31.010 --> 00:39:42.610 Then, this gel needs to be equilibrated in the SDS-PAGE 00:39:42.610 --> 00:39:43.110 buffer. 00:39:50.360 --> 00:39:52.880 And then, you run the SDS-PAGE separation. 00:40:02.120 --> 00:40:05.840 And, in this step, just to note, the gel is rotated 90 degrees. 00:40:16.480 --> 00:40:18.070 OK, so what you get-- 00:40:26.200 --> 00:40:30.330 you get a gel where we have molecular weight here. 00:40:30.330 --> 00:40:33.260 We have pI here. 00:40:33.260 --> 00:40:34.740 And if it's a cell lysate, there's 00:40:34.740 --> 00:40:37.700 going to be many, many spots. 00:40:37.700 --> 00:40:39.720 These should all be spots unless you 00:40:39.720 --> 00:40:41.600 did a poor job running the gel. 00:40:55.680 --> 00:40:58.260 So this 2-D gel is being used, because it's 00:40:58.260 --> 00:41:01.350 going to provide better separation than a standard 1-D 00:41:01.350 --> 00:41:02.040 gel. 00:41:02.040 --> 00:41:05.920 Imagine trying to separate peptides out of some cell 00:41:05.920 --> 00:41:09.330 lysate using just a 1-D gel. 00:41:09.330 --> 00:41:11.220 Even after this immunoprecipitation, 00:41:11.220 --> 00:41:17.400 we'll see that these samples are very complicated here for that. 00:41:17.400 --> 00:41:22.950 So what we need is some way to detect the spots that indicate 00:41:22.950 --> 00:41:24.450 different polypeptides. 00:41:24.450 --> 00:41:26.910 So what are methods? 00:41:26.910 --> 00:41:30.490 Maybe Coomassie stain for total protein. 00:41:30.490 --> 00:41:33.050 We can use the radiolabel-- 00:41:33.050 --> 00:41:36.780 autoradiography, for instance, which is what's done here. 00:41:36.780 --> 00:41:39.480 We're looking at the S35 radiolabel-- 00:41:39.480 --> 00:41:44.350 or maybe Western blot here. 00:41:44.350 --> 00:41:48.570 So how are we going to get from this gel 00:41:48.570 --> 00:41:51.620 to knowing the identity of each of these spots? 00:42:05.850 --> 00:42:07.820 AUDIENCE: You have to identify your spot, 00:42:07.820 --> 00:42:11.300 excise it, extract the protein from the gel, 00:42:11.300 --> 00:42:15.140 adjust it, and then run NS and line it up 00:42:15.140 --> 00:42:17.420 with known protein for evidence. 00:42:17.420 --> 00:42:18.930 ELIZABETH NOLAN: Exactly. 00:42:18.930 --> 00:42:23.780 So what will be done is that each spot of interest 00:42:23.780 --> 00:42:27.260 will be cut out of the gel. 00:42:27.260 --> 00:42:28.760 So you need a way to mark them. 00:42:28.760 --> 00:42:32.690 You'll see they're numbered in the data that we'll look at. 00:42:32.690 --> 00:42:37.220 The protein needs to be extracted out of the gel. 00:42:37.220 --> 00:42:39.920 Then the protein will be incubated 00:42:39.920 --> 00:42:45.530 with a protease that will give some number of fragments. 00:42:45.530 --> 00:42:47.870 Trypsin was used in this work. 00:42:47.870 --> 00:42:50.990 And then that digest can be analyzed by mass spec. 00:42:50.990 --> 00:42:54.920 And so, for each sample, you get all of the m over z values 00:42:54.920 --> 00:42:58.460 for the different polypeptides that resulted from the digest. 00:42:58.460 --> 00:43:00.740 And then, effectively, you can compare that 00:43:00.740 --> 00:43:04.490 to some database of E. coli protein sequences. 00:43:08.020 --> 00:43:12.960 So further details are provided throughout here. 00:43:12.960 --> 00:43:15.610 So what are the major questions? 00:43:15.610 --> 00:43:19.680 And what are we going to look for answers for in the data 00:43:19.680 --> 00:43:20.730 here? 00:43:20.730 --> 00:43:25.740 So first, how many proteins interact with GroEL? 00:43:25.740 --> 00:43:28.260 We can imagine getting an answer to this 00:43:28.260 --> 00:43:31.980 by counting the number of spots. 00:43:31.980 --> 00:43:34.590 What are the identities and structural features 00:43:34.590 --> 00:43:39.240 and properties of the proteins that interact with GroEL? 00:43:39.240 --> 00:43:43.530 We're going to get that from the mass spec analysis and then 00:43:43.530 --> 00:43:45.270 literature studies. 00:43:45.270 --> 00:43:49.260 And then another question we can get at is asking, 00:43:49.260 --> 00:43:51.820 how long do proteins interact with GroEL? 00:43:51.820 --> 00:43:54.870 Because we're calling the pulse-chase samples 00:43:54.870 --> 00:43:56.850 were taken at various time points 00:43:56.850 --> 00:43:59.050 over that 10-minute period. 00:43:59.050 --> 00:44:02.370 So, at two minutes, do we see the same polypeptides 00:44:02.370 --> 00:44:05.700 associated as we see at 10 minutes? 00:44:05.700 --> 00:44:08.880 Or if we monitor one given polypeptide, 00:44:08.880 --> 00:44:10.920 when does it show up and potentially 00:44:10.920 --> 00:44:13.530 disappear from the gels? 00:44:13.530 --> 00:44:18.090 So all of these samples can be addressed with these methods. 00:44:18.090 --> 00:44:21.870 And where we'll begin on Friday is 00:44:21.870 --> 00:44:24.820 going through the data in some detail. 00:44:24.820 --> 00:44:27.700 But just as a prelude to that in the last minute, 00:44:27.700 --> 00:44:30.435 here's the data from the paper for these gels. 00:44:33.960 --> 00:44:40.440 So this is looking at the 2-D gels for, on the top, 00:44:40.440 --> 00:44:44.430 total soluble cytoplasmic proteins at zero minutes 00:44:44.430 --> 00:44:47.220 and then total cytoplasmic proteins at 10 minutes. 00:44:47.220 --> 00:44:50.170 So this is without the immunoprecipitation. 00:44:50.170 --> 00:44:53.250 And then, at the bottom here, what we're looking at 00:44:53.250 --> 00:44:55.980 are the polypeptides that we're isolated 00:44:55.980 --> 00:45:00.840 from the immunoprecipitation with the anti-GroEL antibody 00:45:00.840 --> 00:45:03.360 at zero minutes and 10 minutes. 00:45:03.360 --> 00:45:05.830 And so, before we meet next time, 00:45:05.830 --> 00:45:09.810 what I encourage you to do is take a close look at these gels 00:45:09.810 --> 00:45:13.260 and see what information can you pull out just 00:45:13.260 --> 00:45:15.480 from a qualitative look. 00:45:15.480 --> 00:45:20.983 So simple questions, like, we see a lot of proteins here. 00:45:20.983 --> 00:45:23.150 And please don't go and try and count all the spots. 00:45:23.150 --> 00:45:25.290 I'll give you the numbers next time. 00:45:28.140 --> 00:45:30.870 How do these gels here from the immunoprecipitation 00:45:30.870 --> 00:45:33.090 differ from these up top? 00:45:33.090 --> 00:45:35.220 And it's not just the total number of proteins. 00:45:35.220 --> 00:45:37.880 There's some additional subtleties in these data. 00:45:37.880 --> 00:45:41.370 OK, so next time we'll begin examining these data, 00:45:41.370 --> 00:45:45.150 looking at what polypeptides were pulled down. 00:45:45.150 --> 00:45:50.010 And then we'll move into looking at the chaperone DnaK, 00:45:50.010 --> 00:45:53.360 DnaKJ system there.