1 00:00:00,500 --> 00:00:02,820 The following content is provided under a Creative 2 00:00:02,820 --> 00:00:04,360 Commons license. 3 00:00:04,360 --> 00:00:06,660 Your support will help MIT OpenCourseWare 4 00:00:06,660 --> 00:00:11,020 continue to offer high-quality educational resources for free. 5 00:00:11,020 --> 00:00:13,650 To make a donation or view additional materials 6 00:00:13,650 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,550 at ocw.mit.edu. 8 00:00:25,880 --> 00:00:29,630 ELIZABETH NOLAN: We're going to move on with GroEL/GroES 9 00:00:29,630 --> 00:00:33,860 and a few more comments about where we closed yesterday 10 00:00:33,860 --> 00:00:35,570 and then talk about experiments that 11 00:00:35,570 --> 00:00:37,730 were done to determine what polypeptides 12 00:00:37,730 --> 00:00:40,257 are folded by this machinery. 13 00:00:40,257 --> 00:00:41,090 So I'm just curious. 14 00:00:41,090 --> 00:00:44,390 Has anyone stuck trigger factor or GroEL into PubMed 15 00:00:44,390 --> 00:00:47,070 to see how many hits you get? 16 00:00:47,070 --> 00:00:51,260 Yeah, so Rebecca's question yesterday, or on Monday, 17 00:00:51,260 --> 00:00:54,110 about trigger factor and active versus passive folding 18 00:00:54,110 --> 00:00:56,247 motivated me to take a look. 19 00:00:56,247 --> 00:00:57,830 So just to give you some scope, if you 20 00:00:57,830 --> 00:01:00,110 put trigger factor in PubMed, as of last night, 21 00:01:00,110 --> 00:01:05,860 there's 11,810 hits there. 22 00:01:05,860 --> 00:01:09,650 GroEL is closer to 2,000 to 3,000-- in that range. 23 00:01:09,650 --> 00:01:12,890 If you put trigger factor active folding, 24 00:01:12,890 --> 00:01:15,200 you end up with 34 hits. 25 00:01:15,200 --> 00:01:17,360 Most of those are about using trigger factor 26 00:01:17,360 --> 00:01:19,410 in protein overexpression. 27 00:01:19,410 --> 00:01:23,130 So if you also express trigger factor, does that help? 28 00:01:23,130 --> 00:01:26,240 And it looked like there was one paper in those 34 29 00:01:26,240 --> 00:01:30,980 that suggests an active folding role for one of the domains. 30 00:01:30,980 --> 00:01:33,680 But that is just looking at an abstract. 31 00:01:33,680 --> 00:01:37,760 And so, the point there is there are many, many studies that 32 00:01:37,760 --> 00:01:41,340 consider these chaperones and a huge literature to search. 33 00:01:41,340 --> 00:01:44,420 So what we're able to cover here is really just the tip 34 00:01:44,420 --> 00:01:47,700 of the iceberg for that. 35 00:01:47,700 --> 00:01:51,140 There's also a new review out on GroEL/GroES, 36 00:01:51,140 --> 00:01:52,850 which is not required reading, but we're 37 00:01:52,850 --> 00:01:55,340 posting it on Stellar. 38 00:01:55,340 --> 00:01:57,770 So it just came out last month, and I really 39 00:01:57,770 --> 00:01:59,390 enjoyed reading this review. 40 00:01:59,390 --> 00:02:01,100 I thought they did a very good job 41 00:02:01,100 --> 00:02:06,530 of talking about current questions that are unanswered 42 00:02:06,530 --> 00:02:09,380 yet in terms of models and presenting different models 43 00:02:09,380 --> 00:02:12,560 for how this folding chamber works-- so passive 44 00:02:12,560 --> 00:02:14,760 versus active, for instance. 45 00:02:14,760 --> 00:02:18,620 And they also give a summary of the substrate scope-- 46 00:02:18,620 --> 00:02:21,650 so the experiment we'll talk about today. 47 00:02:21,650 --> 00:02:23,660 So where we left off last time, we 48 00:02:23,660 --> 00:02:27,320 went over the structure of this folding chamber 49 00:02:27,320 --> 00:02:30,570 and here's just another depiction of the overview. 50 00:02:30,570 --> 00:02:35,280 So effectively, we have to back-to-back heptamer rings 51 00:02:35,280 --> 00:02:37,700 as shown here. 52 00:02:37,700 --> 00:02:41,150 Some polypeptide in its non-native state can bind. 53 00:02:41,150 --> 00:02:44,840 It initially binds up at the top by these apical domains, 54 00:02:44,840 --> 00:02:47,250 and there are some hydrophobic interactions. 55 00:02:47,250 --> 00:02:51,250 OK, ATP also binds, and we have all seven ATPs 56 00:02:51,250 --> 00:02:55,970 found within one ring, the ring that has the polypeptide. 57 00:02:55,970 --> 00:02:59,480 OK, we see the lid come on, and then this polypeptide 58 00:02:59,480 --> 00:03:04,340 has some time, a residency time, in this chamber to fold. 59 00:03:04,340 --> 00:03:05,900 And then after the residency time, 60 00:03:05,900 --> 00:03:09,890 which is generally quoted on the order of 6 to 10 seconds, 61 00:03:09,890 --> 00:03:11,790 the lid comes off, and it gets ejected. 62 00:03:11,790 --> 00:03:14,420 And during that time, the ATPs are hydrolyzed. 63 00:03:14,420 --> 00:03:18,770 So somehow, this ATP hydrolysis gives conformational changes 64 00:03:18,770 --> 00:03:20,390 that drive this cycle. 65 00:03:20,390 --> 00:03:23,180 OK, and then we see, again, we flip 66 00:03:23,180 --> 00:03:26,300 to having function in the other ring. 67 00:03:26,300 --> 00:03:29,480 So one point to make involved cooperativity, 68 00:03:29,480 --> 00:03:32,420 so I hope you've all seen cooperativity before, probably 69 00:03:32,420 --> 00:03:34,970 in the context of hemoglobin. 70 00:03:34,970 --> 00:03:38,630 We have examples here of positive cooperativity 71 00:03:38,630 --> 00:03:40,940 and negative cooperativity. 72 00:03:40,940 --> 00:03:46,260 So within one heptamer ring, ATP binds to all seven subunits. 73 00:03:49,070 --> 00:03:51,230 So that's positive cooperativity. 74 00:03:51,230 --> 00:03:53,840 And then we can think about negative cooperativity 75 00:03:53,840 --> 00:03:55,700 between the two rings, where we only 76 00:03:55,700 --> 00:03:57,980 have ATPs bound to one ring. 77 00:03:57,980 --> 00:04:02,300 So the other heptamer ring will not have ATP bound here. 78 00:04:05,250 --> 00:04:09,650 So what is happening inside this chamber? 79 00:04:09,650 --> 00:04:11,990 The polypeptide enters the chamber, 80 00:04:11,990 --> 00:04:15,140 and it's given this protected environment to fold. 81 00:04:15,140 --> 00:04:17,990 And we saw that when the GroES lid comes in 82 00:04:17,990 --> 00:04:21,050 that the hydrophilic nature, hydrophobic nature 83 00:04:21,050 --> 00:04:22,310 of the interior changes. 84 00:04:22,310 --> 00:04:24,770 And it becomes more hydrophilic. 85 00:04:24,770 --> 00:04:28,130 So I just want to point out-- and this also builds upon 86 00:04:28,130 --> 00:04:30,500 Rebecca's question from last time-- 87 00:04:30,500 --> 00:04:32,510 is this passive folding in the chamber 88 00:04:32,510 --> 00:04:34,610 so effectively in Anfinsen's cage, 89 00:04:34,610 --> 00:04:38,870 where the primary sequence dictates the trajectory? 90 00:04:38,870 --> 00:04:42,140 Or does the actual chamber itself play a role? 91 00:04:42,140 --> 00:04:44,540 So that would be active folding. 92 00:04:44,540 --> 00:04:49,580 And effectively, is there forced unfolding or refolding 93 00:04:49,580 --> 00:04:51,440 by GroEL itself? 94 00:04:51,440 --> 00:04:53,960 So perhaps the apical domains can 95 00:04:53,960 --> 00:04:56,450 force unfolding before polypeptide 96 00:04:56,450 --> 00:04:57,890 is released into the chamber. 97 00:04:57,890 --> 00:05:01,340 And the cartoon that was just up indicated that to some degree. 98 00:05:01,340 --> 00:05:05,330 Maybe the cavity walls are involved. 99 00:05:05,330 --> 00:05:07,490 And what I would say is that the pendulum on this 100 00:05:07,490 --> 00:05:10,490 has swayed quite a bit over the years in terms of 101 00:05:10,490 --> 00:05:13,640 whether or not GroEL is a passive folding cage 102 00:05:13,640 --> 00:05:16,340 or actively involved in folding. 103 00:05:16,340 --> 00:05:21,680 And some of the debates in the literature 104 00:05:21,680 --> 00:05:25,430 have resulted from experimental set-up 105 00:05:25,430 --> 00:05:27,770 that may bias results to indicate 106 00:05:27,770 --> 00:05:28,853 one thing or the other. 107 00:05:28,853 --> 00:05:30,770 And that's something the community is striving 108 00:05:30,770 --> 00:05:32,930 to work out these days. 109 00:05:32,930 --> 00:05:35,480 And I'll talk about that a bit more on the next slide. 110 00:05:35,480 --> 00:05:39,710 But I'll just note-- these questions are still there, 111 00:05:39,710 --> 00:05:41,480 and the recent review I just noted 112 00:05:41,480 --> 00:05:44,960 discusses these questions. 113 00:05:44,960 --> 00:05:47,300 There was a study just a few years ago 114 00:05:47,300 --> 00:05:52,310 that was performed with very dilute polypeptide substrate-- 115 00:05:52,310 --> 00:05:54,500 so below one nanomolar. 116 00:05:54,500 --> 00:05:57,500 And what they conclude from this study 117 00:05:57,500 --> 00:06:01,820 is that GroEL is involved in active folding 118 00:06:01,820 --> 00:06:05,200 of a maltose-binding protein mutant. 119 00:06:05,200 --> 00:06:06,950 One question I'll just spring up with this 120 00:06:06,950 --> 00:06:10,790 is, maltose-binding protein is a nice model polypeptide, 121 00:06:10,790 --> 00:06:13,370 but what happens for a native GroEL substrate? 122 00:06:13,370 --> 00:06:16,310 And is there utility in studying those? 123 00:06:16,310 --> 00:06:20,810 So why have I emphasized this dilute protein sample point 124 00:06:20,810 --> 00:06:21,620 here? 125 00:06:21,620 --> 00:06:24,200 So what happened in some early work, 126 00:06:24,200 --> 00:06:26,930 in terms of studies that were done to try to differentiate 127 00:06:26,930 --> 00:06:29,960 active or passive folding, is that there 128 00:06:29,960 --> 00:06:33,780 were some complexities in in vitro studies. 129 00:06:33,780 --> 00:06:39,620 So, here, I just have a cartoon of folding in the chamber. 130 00:06:39,620 --> 00:06:43,340 And if we think about only one polypeptide within the GroEL 131 00:06:43,340 --> 00:06:46,190 chamber, it's folding in isolation. 132 00:06:46,190 --> 00:06:48,380 So there's no possibility for it to form 133 00:06:48,380 --> 00:06:52,360 an aggregate or a ligamer with other polypeptides. 134 00:06:52,360 --> 00:06:55,070 It's all alone here. 135 00:06:55,070 --> 00:06:58,100 So this folding in the chamber avoids the complications 136 00:06:58,100 --> 00:06:59,600 of the folding landscape we talked 137 00:06:59,600 --> 00:07:02,780 about in the introductory lecture to this module. 138 00:07:02,780 --> 00:07:07,060 So what happens in aqueous solution, right? 139 00:07:07,060 --> 00:07:10,400 There's the possibility that, depending on your conditions, 140 00:07:10,400 --> 00:07:13,460 maybe there's some sort of aggregate that forms. 141 00:07:13,460 --> 00:07:15,350 And if this aggregate forms, what does that 142 00:07:15,350 --> 00:07:18,170 mean in terms of what you see? 143 00:07:18,170 --> 00:07:21,650 And so, in earlier work, there were 144 00:07:21,650 --> 00:07:24,170 some in vitro kinetic studies that 145 00:07:24,170 --> 00:07:27,860 indicated GroEL accelerates folding relative to folding 146 00:07:27,860 --> 00:07:30,530 in dilute aqueous solution. 147 00:07:30,530 --> 00:07:33,110 But some of these comparisons weren't appropriate, 148 00:07:33,110 --> 00:07:38,420 because as it turns out, oligomerization might compete 149 00:07:38,420 --> 00:07:40,280 with what you're watching for. 150 00:07:40,280 --> 00:07:43,760 And so, if there's some oligomerization happening, 151 00:07:43,760 --> 00:07:46,910 it might indicate that the rate is slower than you think. 152 00:07:46,910 --> 00:07:48,590 So there's ways to monitor for this. 153 00:07:48,590 --> 00:07:52,040 And it's just a point in terms of what control studies do 154 00:07:52,040 --> 00:07:55,220 you need to do to make sure your experimental setup is 155 00:07:55,220 --> 00:07:57,585 appropriate there. 156 00:07:57,585 --> 00:07:59,210 I think it'll be exciting to see what's 157 00:07:59,210 --> 00:08:02,540 to come in future years about this question 158 00:08:02,540 --> 00:08:05,390 and what kinds of biophysical techniques 159 00:08:05,390 --> 00:08:10,610 are applied, including single-molecule studies here. 160 00:08:10,610 --> 00:08:13,340 So where we're going to go, moving on, 161 00:08:13,340 --> 00:08:17,810 is to think about what actually are the substrates for GroEL. 162 00:08:17,810 --> 00:08:21,200 So what polypeptides get folded in this chamber? 163 00:08:21,200 --> 00:08:24,170 And how do we begin to address that question 164 00:08:24,170 --> 00:08:27,410 from the standpoint of what's happening in the cell? 165 00:08:27,410 --> 00:08:31,220 OK, so first, we're just going to consider some observations. 166 00:08:31,220 --> 00:08:34,580 And then we're going to go into the experiments here. 167 00:08:34,580 --> 00:08:37,610 So here are some observations. 168 00:08:37,610 --> 00:08:40,010 So the first one is that polypeptides, 169 00:08:40,010 --> 00:08:44,150 up to 60 kilodaltons, can fold in this chamber. 170 00:08:44,150 --> 00:08:45,620 So that's quite big-- 171 00:08:45,620 --> 00:08:48,560 60 kilodaltons. 172 00:08:48,560 --> 00:08:51,380 Some proteins or polypeptides need 173 00:08:51,380 --> 00:08:55,550 to enter the GroEL multiple times to be folded. 174 00:08:55,550 --> 00:08:59,270 So that means the chaperone has the ability to bind and release 175 00:08:59,270 --> 00:09:03,470 and re-bind the polypeptide here. 176 00:09:03,470 --> 00:09:07,370 So when studies are done in vitro, what's found 177 00:09:07,370 --> 00:09:11,750 is that almost all polypeptides interact with GroEL. 178 00:09:11,750 --> 00:09:15,320 So you just saw even an example of that 179 00:09:15,320 --> 00:09:18,020 in terms of this non-native maltose-binding protein. 180 00:09:18,020 --> 00:09:20,720 So many polypeptides will interact. 181 00:09:20,720 --> 00:09:24,140 And this really contrasts what's observed in the cell, 182 00:09:24,140 --> 00:09:30,455 where, in vivo GroEL is involved in only folding about 10% of E. 183 00:09:30,455 --> 00:09:32,510 coli proteins here. 184 00:09:32,510 --> 00:09:36,650 OK, so what observations three and four suggest 185 00:09:36,650 --> 00:09:39,590 is that GroEL has some preference 186 00:09:39,590 --> 00:09:42,680 for particular endogenous polypeptides. 187 00:09:42,680 --> 00:09:46,640 And what we want to answer is, what are these polypeptides, 188 00:09:46,640 --> 00:09:49,160 and what are their properties here? 189 00:09:49,160 --> 00:09:52,670 OK, so Hartl's group did some nice studies 190 00:09:52,670 --> 00:09:56,138 to look at this, what needs to be done. 191 00:09:56,138 --> 00:09:57,680 First of all, there needs to be a way 192 00:09:57,680 --> 00:10:00,710 to isolate the polypeptides that are interacting 193 00:10:00,710 --> 00:10:03,260 with GroEL in the cell. 194 00:10:03,260 --> 00:10:06,290 And then, once these polypeptides are isolated, 195 00:10:06,290 --> 00:10:08,450 they need to be analyzed in order 196 00:10:08,450 --> 00:10:11,640 to learn about their identity and properties. 197 00:10:11,640 --> 00:10:14,120 OK, so we're going to look at experiments 198 00:10:14,120 --> 00:10:17,180 that were done to address this. 199 00:10:17,180 --> 00:10:20,120 And they involve pulse-chase labeling 200 00:10:20,120 --> 00:10:24,290 of newly synthesized proteins, amino precipitation, 201 00:10:24,290 --> 00:10:25,860 and analysis here. 202 00:10:25,860 --> 00:10:30,502 So in terms of addressing what are these substrates, 203 00:10:30,502 --> 00:10:32,460 we're going to begin with pulse-chase labeling. 204 00:10:42,980 --> 00:10:47,885 OK, so basically, the goal of this experiment 205 00:10:47,885 --> 00:10:49,260 and why we're starting here is we 206 00:10:49,260 --> 00:11:06,740 want to determine which proteins interact with GroEL. 207 00:11:06,740 --> 00:11:08,870 And, in addition to which proteins, 208 00:11:08,870 --> 00:11:10,850 we want to determine how long they interact. 209 00:11:27,946 --> 00:11:31,320 OK, so what is the experiment? 210 00:11:31,320 --> 00:11:32,880 These experiments are going to be 211 00:11:32,880 --> 00:11:36,080 done with like E. coli cells. 212 00:11:36,080 --> 00:11:38,220 So we want to know what's happening in the cell. 213 00:11:38,220 --> 00:11:39,750 So imagine we have an E. coli. 214 00:11:43,250 --> 00:11:47,490 And so these bacteria are grown in some culture medium. 215 00:11:47,490 --> 00:11:49,110 And the trick here is that they're 216 00:11:49,110 --> 00:11:55,140 going to be grown in medium that's depleted in methionine. 217 00:11:55,140 --> 00:12:11,350 So incubate, or grow, in medium with no methionine. 218 00:12:11,350 --> 00:12:16,850 OK, so effectively, we're depleting them 219 00:12:16,850 --> 00:12:19,610 of that amino acid. 220 00:12:19,610 --> 00:12:23,320 OK, so then after some period of growth, 221 00:12:23,320 --> 00:12:25,040 what are we going to do? 222 00:12:25,040 --> 00:12:29,440 We're going to spike the culture with radiolabeled methionine. 223 00:12:29,440 --> 00:12:31,720 And this is the pulse. 224 00:12:31,720 --> 00:12:35,860 So we're going to add 35S methionine. 225 00:12:38,950 --> 00:12:42,600 And we're then going to incubate for 15 seconds. 226 00:12:46,650 --> 00:12:51,330 OK, and so that's the pulse with a radiolabeled amino acid. 227 00:12:51,330 --> 00:12:52,650 Then what are we going to do? 228 00:12:52,650 --> 00:12:56,550 And after we go through the steps, we'll go through why. 229 00:12:56,550 --> 00:12:58,980 After this stage, we're going to add 230 00:12:58,980 --> 00:13:01,300 excess unlabeled methionine. 231 00:13:13,980 --> 00:13:19,900 And we're going to then continue this culture for 10 minutes. 232 00:13:19,900 --> 00:13:24,690 OK, this is the chase here. 233 00:13:24,690 --> 00:13:28,290 And during this chase period, basically, samples 234 00:13:28,290 --> 00:13:35,765 will be taken at varying time points. 235 00:13:40,060 --> 00:13:42,910 OK, and then, at some point, we're just going to stop this. 236 00:13:42,910 --> 00:13:50,760 OK, so just say, stop culture and experiment. 237 00:13:56,270 --> 00:14:00,530 So what's happening in each of these steps? 238 00:14:00,530 --> 00:14:02,990 And why are we doing this? 239 00:14:02,990 --> 00:14:09,530 So what we want to do is think about newly translated 240 00:14:09,530 --> 00:14:11,030 polypeptides. 241 00:14:11,030 --> 00:14:13,730 OK, so we have a living E. coli. 242 00:14:13,730 --> 00:14:14,750 It has ribosomes. 243 00:14:14,750 --> 00:14:17,990 And these ribosomes are going to be synthesizing polypeptides 244 00:14:17,990 --> 00:14:21,210 over the course of this experiment. 245 00:14:21,210 --> 00:14:35,850 So during the pulse period, all proteins, or all polypeptides, 246 00:14:35,850 --> 00:14:42,090 synthesized are radiolabeled. 247 00:14:51,210 --> 00:14:53,840 Right, because the methionine has been depleted 248 00:14:53,840 --> 00:14:55,580 from the culture medium. 249 00:14:55,580 --> 00:14:57,920 And so effectively, the methionine 250 00:14:57,920 --> 00:15:03,900 that these organisms are seeing are the S35-labeled methionine. 251 00:15:03,900 --> 00:15:07,970 And all polypeptides have an informal methionine 252 00:15:07,970 --> 00:15:11,120 from the initiator tRNA and what other methionines 253 00:15:11,120 --> 00:15:13,310 are in the sequence. 254 00:15:13,310 --> 00:15:17,330 So, if we think about doing this for 15 seconds, 255 00:15:17,330 --> 00:15:21,130 and we think about the translation rate, which 256 00:15:21,130 --> 00:15:24,260 I gave as 6 to 20 amino acids per second 257 00:15:24,260 --> 00:15:26,690 when we were discussing the ribosome, 258 00:15:26,690 --> 00:15:30,710 we want to think about how long are these polypeptides going 259 00:15:30,710 --> 00:15:31,370 to be? 260 00:15:31,370 --> 00:15:39,530 So we have a translation rate of 6 to 20 amino acids per second. 261 00:15:39,530 --> 00:15:45,260 OK, so, if we think about 15 seconds of a pulse, 262 00:15:45,260 --> 00:15:48,320 we're getting polypeptides on the order 263 00:15:48,320 --> 00:15:56,800 of 90 to 300 amino acids synthesized during that time. 264 00:16:00,850 --> 00:16:05,080 So newly synthesized polypeptides in these 15 265 00:16:05,080 --> 00:16:07,030 seconds are radiolabeled. 266 00:16:07,030 --> 00:16:09,190 What happens next? 267 00:16:09,190 --> 00:16:12,730 OK, we have this chase period where we flood the system 268 00:16:12,730 --> 00:16:14,860 with unlabeled methionine here. 269 00:16:24,160 --> 00:16:27,640 Why are we doing this? 270 00:16:27,640 --> 00:16:30,280 So certainly, there are some polypeptides 271 00:16:30,280 --> 00:16:35,320 that are longer than 300 amino acids that still need time 272 00:16:35,320 --> 00:16:37,120 to be synthesized. 273 00:16:37,120 --> 00:16:41,260 And if there's new peptides being synthesized 274 00:16:41,260 --> 00:16:43,690 that start in this stage, we won't see them, 275 00:16:43,690 --> 00:16:45,470 because this unlabeled methionine 276 00:16:45,470 --> 00:16:49,210 is in vast access over the radiolabeled methionine that 277 00:16:49,210 --> 00:16:52,060 was added early. 278 00:16:52,060 --> 00:17:07,920 So here, we have, the synthesis of larger polypeptides 279 00:17:07,920 --> 00:17:08,970 can be completed. 280 00:17:20,900 --> 00:17:35,840 And we have, no longer producing radiolabeled new peptides. 281 00:17:41,910 --> 00:17:44,880 OK, so this allows us to only see 282 00:17:44,880 --> 00:17:47,550 the peptides that were radiolabeled during this pulse 283 00:17:47,550 --> 00:17:48,690 period here. 284 00:17:55,160 --> 00:17:57,230 So what are we going to do in terms 285 00:17:57,230 --> 00:18:00,710 of the sampling at various time points? 286 00:18:08,900 --> 00:18:12,800 So let's say we want to sample at one minute, five minutes, 287 00:18:12,800 --> 00:18:13,490 ten minutes. 288 00:18:13,490 --> 00:18:15,330 What do we need to do? 289 00:18:15,330 --> 00:18:18,020 So can we just aliquot some of these E. coli 290 00:18:18,020 --> 00:18:19,895 and put them on our bench? 291 00:18:32,570 --> 00:18:37,050 We could, but that's not going to be very helpful to us, 292 00:18:37,050 --> 00:18:40,910 because what we want to do is stop the translation 293 00:18:40,910 --> 00:18:45,325 machinery and all of the cellular machinery here. 294 00:18:45,325 --> 00:18:46,950 AUDIENCE: You need some kind of clench? 295 00:18:46,950 --> 00:18:48,980 ELIZABETH NOLAN: Yeah, we need a clench. 296 00:18:48,980 --> 00:18:50,810 And not only do we need a clench, 297 00:18:50,810 --> 00:18:53,600 we're dealing with a living organism too, right? 298 00:18:53,600 --> 00:18:57,980 So we need to break open the E. coli in whatever this condition 299 00:18:57,980 --> 00:19:00,320 is to stop the reaction. 300 00:19:00,320 --> 00:19:10,208 OK, so we're going to take aliquots 301 00:19:10,208 --> 00:19:11,700 at varying time points. 302 00:19:16,150 --> 00:19:18,940 And basically, we care about time, 303 00:19:18,940 --> 00:19:22,120 so you have to immediately lyse, or break open, the cells. 304 00:19:27,190 --> 00:19:29,575 And this was done in the presence of EDTA. 305 00:19:39,040 --> 00:19:39,978 So what is EDTA? 306 00:19:39,978 --> 00:19:41,478 AUDIENCE: Ethylenediaminetetraacetic 307 00:19:41,478 --> 00:19:41,978 acid. 308 00:19:41,978 --> 00:19:44,570 ELIZABETH NOLAN: Yeah, ethylenediaminetetraacetic 309 00:19:44,570 --> 00:19:45,070 acid. 310 00:19:45,070 --> 00:19:47,890 So it's the chelator. 311 00:19:47,890 --> 00:19:52,300 And why might this lysis be done in the presence of this metal 312 00:19:52,300 --> 00:19:54,604 chelator? 313 00:19:54,604 --> 00:19:59,810 AUDIENCE: [INAUDIBLE] processes like-- 314 00:19:59,810 --> 00:20:05,330 [INAUDIBLE] magnesium, which would help [INAUDIBLE] 315 00:20:05,330 --> 00:20:10,520 AUDIENCE: Are the proteases that are not binding? 316 00:20:10,520 --> 00:20:12,890 ELIZABETH NOLAN: There certainly are zinc proteases. 317 00:20:12,890 --> 00:20:16,610 So that that's one class of protease. 318 00:20:16,610 --> 00:20:21,890 So EDTA will chelate many, many different metals. 319 00:20:21,890 --> 00:20:26,180 The main point here is we want to stop stop translation, 320 00:20:26,180 --> 00:20:29,470 shut down processes here. 321 00:20:29,470 --> 00:20:32,665 OK, so we have these samples. 322 00:20:35,470 --> 00:20:36,685 What do we need to do next? 323 00:20:47,560 --> 00:21:06,370 We need to detect these newly synthesized proteins that 324 00:21:06,370 --> 00:21:14,200 interact with GroEL. 325 00:21:14,200 --> 00:21:17,430 And we want to do this at each time point. 326 00:21:17,430 --> 00:21:19,200 So how are we going to do this? 327 00:21:19,200 --> 00:21:22,550 We have a very complex mixture that has 328 00:21:22,550 --> 00:21:24,440 all of the cellular components. 329 00:21:36,210 --> 00:21:41,330 So the next step in this will be immunoprecipitation. 330 00:21:54,850 --> 00:21:58,270 And so, what will happen in immunoprecipitation 331 00:21:58,270 --> 00:22:02,470 in these experiments is that the researchers had an antibody 332 00:22:02,470 --> 00:22:04,210 that binds to GroEL. 333 00:22:04,210 --> 00:22:07,600 And this antibody was put on a bead 334 00:22:07,600 --> 00:22:12,040 and used to fish out GroEL from this complex mixture. 335 00:22:12,040 --> 00:22:15,190 And we need to talk about these antibodies a little more. 336 00:22:15,190 --> 00:22:20,560 But just in starting, I imagine there's a bead. 337 00:22:20,560 --> 00:22:26,870 And we think about antibodies as being Y-shaped biomolecules. 338 00:22:26,870 --> 00:22:28,030 So here, we have a GroEL. 339 00:22:37,550 --> 00:22:41,240 And imagine that, in this mixture, 340 00:22:41,240 --> 00:22:46,430 we have GroEL that has some polypeptide bound. 341 00:22:52,810 --> 00:22:56,260 That's one of its endogenous substrates. 342 00:22:56,260 --> 00:23:05,420 So, if these are mixed together, then the antibody 343 00:23:05,420 --> 00:23:12,765 binds GroEL with the polypeptide attached. 344 00:23:16,620 --> 00:23:24,760 OK, here, we can imagine "capture" of this species 345 00:23:24,760 --> 00:23:29,500 here and using the bead to separate, say, 346 00:23:29,500 --> 00:23:32,230 by centrifugation. 347 00:23:32,230 --> 00:23:36,780 So let's think about this a little bit 348 00:23:36,780 --> 00:23:40,750 and a little background to have everyone up to speed. 349 00:23:40,750 --> 00:23:43,240 If you need to learn more about antibodies, 350 00:23:43,240 --> 00:23:47,710 please see a basic biology textbook for further details. 351 00:23:47,710 --> 00:23:49,870 But these are Y-shaped molecules that 352 00:23:49,870 --> 00:23:53,710 are produced by a type of immune cell called B cells. 353 00:23:53,710 --> 00:23:55,870 And they're used by the immune system 354 00:23:55,870 --> 00:24:01,270 to detect foreign biomolecules and help to neutralize them. 355 00:24:01,270 --> 00:24:03,550 And so, in these, the tip of the Y 356 00:24:03,550 --> 00:24:07,990 contains the paratope that ideally binds specifically 357 00:24:07,990 --> 00:24:12,850 to a particular epitope-- in this case, GroEL here. 358 00:24:12,850 --> 00:24:15,880 And so, we often think about a lock-and-key model 359 00:24:15,880 --> 00:24:19,570 with antibody and think about the antibody binding its target 360 00:24:19,570 --> 00:24:24,140 with precision here. 361 00:24:24,140 --> 00:24:27,250 So for these experiments that were done, 362 00:24:27,250 --> 00:24:29,410 just realize the researchers had to come up 363 00:24:29,410 --> 00:24:31,510 with an antibody to GroEL. 364 00:24:31,510 --> 00:24:33,040 How is that done? 365 00:24:33,040 --> 00:24:36,390 They may have immunized, say, a rabbit 366 00:24:36,390 --> 00:24:39,910 or given a rabbit GroEL and allowed that rabbit 367 00:24:39,910 --> 00:24:40,990 to produce antibodies. 368 00:24:40,990 --> 00:24:44,110 And then they isolate the antibodies here. 369 00:24:44,110 --> 00:24:47,650 So something we want you to take home from this course 370 00:24:47,650 --> 00:24:51,890 is, yes, the antibodies should bind the target with precision. 371 00:24:51,890 --> 00:24:54,790 But there's huge problems in terms of use 372 00:24:54,790 --> 00:24:57,040 of antibodies in research. 373 00:24:57,040 --> 00:24:59,800 This is just the start of an article that was published 374 00:24:59,800 --> 00:25:01,270 last year around this time. 375 00:25:01,270 --> 00:25:03,670 And it's focused on pharma and clinical trials. 376 00:25:03,670 --> 00:25:05,890 But this is much more broad. 377 00:25:05,890 --> 00:25:09,850 And often, antibodies aren't as specific as indicated 378 00:25:09,850 --> 00:25:15,130 by the label on the container from the supplier here. 379 00:25:15,130 --> 00:25:16,960 And it's pretty dismal what they quote 380 00:25:16,960 --> 00:25:22,810 in this terms of how difficult it is to reproduce data here. 381 00:25:22,810 --> 00:25:24,910 So if you're going to use an antibody, 382 00:25:24,910 --> 00:25:30,100 you always need to test it to see whether it is selective 383 00:25:30,100 --> 00:25:33,040 or not for the species of interest 384 00:25:33,040 --> 00:25:35,740 that you want to detect there and have 385 00:25:35,740 --> 00:25:39,490 that information on hand so you don't misinterpret your data 386 00:25:39,490 --> 00:25:43,060 here for that. 387 00:25:43,060 --> 00:25:48,430 So what are the steps for this immunoprecipitation? 388 00:25:48,430 --> 00:25:51,910 Basically, as shown on the board, 389 00:25:51,910 --> 00:25:54,670 beads will be functionalized with the antibody 390 00:25:54,670 --> 00:25:58,330 and then just added to the cell lysate. 391 00:25:58,330 --> 00:26:03,250 And the antibody can recognize GroEL. 392 00:26:03,250 --> 00:26:06,970 And the goal and hope are that whatever 393 00:26:06,970 --> 00:26:11,590 polypeptides are associated with GroEL are pulled down together. 394 00:26:11,590 --> 00:26:13,090 So that's something a bit incredible 395 00:26:13,090 --> 00:26:17,260 here that these polypeptides remain bound to GroEL 396 00:26:17,260 --> 00:26:19,900 during the steps of this process. 397 00:26:19,900 --> 00:26:22,360 You can imagine, if there's a low-affinity binder, 398 00:26:22,360 --> 00:26:23,890 it could be lost. 399 00:26:23,890 --> 00:26:26,200 So the sample can be centrifuged. 400 00:26:26,200 --> 00:26:28,810 And then, you can isolate these beads here. 401 00:26:31,450 --> 00:26:34,780 So, in cartoon form, a complex cell 402 00:26:34,780 --> 00:26:37,680 lysate in your microcentrifuge tube. 403 00:26:37,680 --> 00:26:41,710 You can add the antibody, centrifuge. 404 00:26:41,710 --> 00:26:44,170 And see, down here, we've pelleted the beads 405 00:26:44,170 --> 00:26:45,820 with GroEL attached. 406 00:26:45,820 --> 00:26:47,440 And then some sort of workup needs 407 00:26:47,440 --> 00:26:51,640 to be done to dissociate the protein, or polypeptide, 408 00:26:51,640 --> 00:26:52,960 substrates here. 409 00:26:52,960 --> 00:26:55,432 And then they can be analyzed. 410 00:26:55,432 --> 00:26:58,258 AUDIENCE: How long do they do that for? 411 00:26:58,258 --> 00:26:59,680 Do you know how many-- 412 00:26:59,680 --> 00:27:01,722 ELIZABETH NOLAN: How long do they centrifuge for? 413 00:27:01,722 --> 00:27:04,631 AUDIENCE: No, for the immunoprecipitation. 414 00:27:04,631 --> 00:27:05,570 Is it 30 minutes? 415 00:27:05,570 --> 00:27:06,070 Is it-- 416 00:27:06,070 --> 00:27:07,870 ELIZABETH NOLAN: I don't know how long the incubation 417 00:27:07,870 --> 00:27:08,370 time is. 418 00:27:08,370 --> 00:27:09,940 Need to go back to the experimental, 419 00:27:09,940 --> 00:27:13,180 but that's getting right back to this question as to how 420 00:27:13,180 --> 00:27:14,210 do they stay bound. 421 00:27:14,210 --> 00:27:15,708 AUDIENCE: How do they stay bound? 422 00:27:15,708 --> 00:27:16,625 ELIZABETH NOLAN: Yeah. 423 00:27:20,530 --> 00:27:21,490 So, see the point here. 424 00:27:21,490 --> 00:27:24,310 If you have a high-affinity complex, that's one thing. 425 00:27:24,310 --> 00:27:26,840 If you have low-affinity association between GroEL 426 00:27:26,840 --> 00:27:29,080 and the polypeptide, you can imagine 427 00:27:29,080 --> 00:27:32,360 it might get lost during this workup. 428 00:27:32,360 --> 00:27:37,240 And how much do we know about those affinities there? 429 00:27:37,240 --> 00:27:40,400 AUDIENCE: You said that they would just give rabbits GroEL, 430 00:27:40,400 --> 00:27:44,500 and hopefully antibodies would just happen. 431 00:27:44,500 --> 00:27:47,810 But if a rabbit's immune system encountered GroEL, 432 00:27:47,810 --> 00:27:50,884 would it actually see it as an antigen 433 00:27:50,884 --> 00:27:53,630 that it had to develop antibodies against? 434 00:27:53,630 --> 00:27:55,030 ELIZABETH NOLAN: So, yeah. 435 00:27:55,030 --> 00:27:56,500 So here's the point-- 436 00:27:56,500 --> 00:27:58,150 would it? 437 00:27:58,150 --> 00:28:03,880 So, if it's E. coli GroEL, would the rabbit recognize this, 438 00:28:03,880 --> 00:28:04,660 yes or no? 439 00:28:04,660 --> 00:28:08,200 And if no, then what can you do to provoke an antibody 440 00:28:08,200 --> 00:28:08,910 response? 441 00:28:08,910 --> 00:28:10,630 And so, what can be done is, say, 442 00:28:10,630 --> 00:28:13,990 you could take a GroEL subunit and attach that 443 00:28:13,990 --> 00:28:15,970 to something immunogenic. 444 00:28:15,970 --> 00:28:18,520 So there are carrier proteins that 445 00:28:18,520 --> 00:28:21,100 will mount an immune response. 446 00:28:21,100 --> 00:28:25,120 So one of the subunits of cholera toxin 447 00:28:25,120 --> 00:28:27,490 is an example that can be used. 448 00:28:27,490 --> 00:28:29,920 And then the idea is you're mounting an immune response 449 00:28:29,920 --> 00:28:31,270 against that carrier protein. 450 00:28:31,270 --> 00:28:35,170 But you'll also get antibodies to whatever is attached. 451 00:28:35,170 --> 00:28:37,480 So that's another strategy for doing it 452 00:28:37,480 --> 00:28:39,370 if direct injection doesn't work. 453 00:28:39,370 --> 00:28:42,790 And too, not going off on a big tangent, 454 00:28:42,790 --> 00:28:45,290 but there are some decisions that need to be made. 455 00:28:45,290 --> 00:28:47,920 So would they use the full-length GroEL? 456 00:28:47,920 --> 00:28:51,160 Or maybe they would just use a polypeptide region, 457 00:28:51,160 --> 00:28:55,060 like some shorter polypeptide that's a portion of GroEL. 458 00:28:55,060 --> 00:28:56,710 So there's a lot of possibilities 459 00:28:56,710 --> 00:28:58,960 there in terms of what you use to generate 460 00:28:58,960 --> 00:29:06,190 the antibody for that there. 461 00:29:06,190 --> 00:29:10,930 And it's something that a lot of companies do these days. 462 00:29:10,930 --> 00:29:15,490 You can send them your protein or your polypeptide fragment. 463 00:29:15,490 --> 00:29:19,150 And they'll conjugate it to one of these carriers 464 00:29:19,150 --> 00:29:23,590 and treat the rabbits or whatever animal 465 00:29:23,590 --> 00:29:25,540 and then isolate those antibodies. 466 00:29:25,540 --> 00:29:29,770 And then they need to be characterized there for that. 467 00:29:29,770 --> 00:29:36,910 OK, so how are these samples going to be analyzed? 468 00:29:36,910 --> 00:29:38,710 That's the next step. 469 00:29:41,320 --> 00:29:45,810 So, for the analysis, effectively, we're 470 00:29:45,810 --> 00:29:48,010 going to have some mixture. 471 00:29:48,010 --> 00:29:50,440 And, at the onset, we don't really 472 00:29:50,440 --> 00:29:54,340 know how complicated this mixture will be. 473 00:29:54,340 --> 00:29:59,440 I told you initially that about 10% of E. coli polypeptides 474 00:29:59,440 --> 00:30:02,470 are thought to be substrates for GroEL, which 475 00:30:02,470 --> 00:30:04,990 is quite a large number if we think about the total number 476 00:30:04,990 --> 00:30:07,840 of proteins in E. coli. 477 00:30:07,840 --> 00:30:11,020 And the other point is we have this radiolabel, which 478 00:30:11,020 --> 00:30:14,110 we're going to use for detection there. 479 00:30:14,110 --> 00:30:16,750 OK, so, for analysis-- 480 00:30:35,530 --> 00:30:36,590 OK, there's two things. 481 00:30:36,590 --> 00:30:40,100 We need to separate these various polypeptides 482 00:30:40,100 --> 00:30:42,230 in each sample. 483 00:30:42,230 --> 00:30:45,770 And then we need to determine what their identities are here. 484 00:30:48,480 --> 00:31:16,855 So-- that were bound to GroEL from one another. 485 00:31:23,450 --> 00:31:28,400 OK, and then, we need to determine identities. 486 00:31:32,480 --> 00:31:34,070 And once we know the identities, we 487 00:31:34,070 --> 00:31:36,590 can think about their properties. 488 00:31:36,590 --> 00:31:39,770 And this needs to be done in every sample that was collected 489 00:31:39,770 --> 00:31:43,460 along this time course, which is also going to give 490 00:31:43,460 --> 00:31:45,870 some temporal information. 491 00:31:45,870 --> 00:31:48,870 So what are the methods that have been used? 492 00:31:48,870 --> 00:31:51,770 So, in order to separate the proteins in this complex 493 00:31:51,770 --> 00:31:56,090 sample, the method is a 2-D gel-- 494 00:31:56,090 --> 00:31:58,730 so 2-D gel electrophoresis. 495 00:32:06,500 --> 00:32:11,910 OK, and in terms of determining the identities, what's 496 00:32:11,910 --> 00:32:14,890 done, once these polypeptides are separated, 497 00:32:14,890 --> 00:32:23,260 is to do a protease digest and then mass spectrometry. 498 00:32:31,950 --> 00:32:40,540 Has anyone here ever run a 2-D gel or seen the equipment? 499 00:32:40,540 --> 00:32:41,860 One person. 500 00:32:41,860 --> 00:32:44,816 Has anyone heard of 2-D gels? 501 00:32:44,816 --> 00:32:45,940 Fair number. 502 00:32:45,940 --> 00:32:50,950 OK, so, we'll go over this briefly in terms of 2-D gel. 503 00:33:11,060 --> 00:33:23,090 So, in terms of 2-D gel electrophoresis, 504 00:33:23,090 --> 00:33:27,140 we talk about running these gels in two dimensions. 505 00:33:27,140 --> 00:33:29,540 And, in each dimension, we separate 506 00:33:29,540 --> 00:33:31,490 based on a different property. 507 00:33:31,490 --> 00:33:39,720 So, in the first dimension, the separation is based on charge. 508 00:33:52,400 --> 00:33:59,670 And effectively, we can talk about the pI of a protein. 509 00:33:59,670 --> 00:34:04,240 So the pI is the isoelectric point. 510 00:34:09,820 --> 00:34:13,960 And it's the pH where the net charge on the protein is zero. 511 00:34:27,500 --> 00:34:31,550 And so, the type of gel we use here 512 00:34:31,550 --> 00:34:35,869 is called isoelectric focusing, or IEF. 513 00:34:48,920 --> 00:34:54,920 And effectively, what's done is that the gel electrophoresis is 514 00:34:54,920 --> 00:34:57,410 done through a continuous and stable pH gradient. 515 00:35:27,190 --> 00:35:31,720 And, in this gel, the protein will migrate to a position 516 00:35:31,720 --> 00:35:33,560 where the pH corresponds to the pI. 517 00:35:37,140 --> 00:35:39,955 Then the anode is low pH and the cathode high pH. 518 00:35:43,780 --> 00:35:48,130 So that's quite different than SDS, where, in an SDS-PAGE gel, 519 00:35:48,130 --> 00:35:53,230 we're coating the protein with negative charge. 520 00:35:53,230 --> 00:36:00,340 So then, the second dimension is something most of us 521 00:36:00,340 --> 00:36:02,040 are familiar with, is SDS-PAGE. 522 00:36:08,810 --> 00:36:14,150 And so, what happens in SDS-PAGE? 523 00:36:14,150 --> 00:36:24,490 We have separation based on size here-- 524 00:36:27,850 --> 00:36:29,140 on molecular weight. 525 00:36:34,280 --> 00:36:38,270 So has anyone not run an SDS-PAGE gel? 526 00:36:38,270 --> 00:36:39,560 And this is totally fine. 527 00:36:39,560 --> 00:36:41,200 I never ran one till I was a postdoc. 528 00:36:41,200 --> 00:36:45,390 So it's not something to be ashamed about if you haven't. 529 00:36:45,390 --> 00:36:46,750 OK, so everyone has. 530 00:36:46,750 --> 00:36:51,100 So what's the ratio of SDS molecules to amino acids? 531 00:36:54,110 --> 00:36:55,960 So if you take your protein sample 532 00:36:55,960 --> 00:37:02,582 and you put it in your loading buffer and run your SDS-PAGE, 533 00:37:02,582 --> 00:37:03,790 what is the ratio of binding? 534 00:37:14,650 --> 00:37:16,642 What is SDS? 535 00:37:16,642 --> 00:37:20,050 AUDIENCE: Sodium dodecyl sulfate. 536 00:37:20,050 --> 00:37:21,670 ELIZABETH NOLAN: And what does it do? 537 00:37:21,670 --> 00:37:23,990 What happens to your protein in SDS? 538 00:37:23,990 --> 00:37:25,250 AUDIENCE: Denatures it. 539 00:37:25,250 --> 00:37:26,542 ELIZABETH NOLAN: OK, what else? 540 00:37:26,542 --> 00:37:27,430 So it's a denaturant. 541 00:37:27,430 --> 00:37:28,597 So it denatures the protein. 542 00:37:33,120 --> 00:37:35,070 So why does SDS-PAGE let you separate 543 00:37:35,070 --> 00:37:37,210 based on molecular weight, more or less? 544 00:37:37,210 --> 00:37:39,720 AUDIENCE: It coats the protein, more or less, 545 00:37:39,720 --> 00:37:41,983 uniformly with negative charge. 546 00:37:41,983 --> 00:37:42,900 ELIZABETH NOLAN: Yeah. 547 00:37:42,900 --> 00:37:45,312 AUDIENCE: Do we know the exact ratio of binding? 548 00:37:45,312 --> 00:37:47,520 ELIZABETH NOLAN: Yeah, so what's the ratio of binding 549 00:37:47,520 --> 00:37:50,370 that can be done in terms of grams 550 00:37:50,370 --> 00:37:53,880 of SDS per grams of protein or number of SDS 551 00:37:53,880 --> 00:37:56,490 molecules per amino acid. 552 00:37:56,490 --> 00:37:59,330 What is it? 553 00:37:59,330 --> 00:38:01,832 And there'll be some error, but there's approximates. 554 00:38:01,832 --> 00:38:03,540 But it's something to think about, right? 555 00:38:03,540 --> 00:38:06,120 You're putting your sample into this. 556 00:38:06,120 --> 00:38:11,010 So it's about 1.4 grams of SDS per gram of protein. 557 00:38:11,010 --> 00:38:13,470 That's the ratio there. 558 00:38:13,470 --> 00:38:18,360 And as said, the idea is that SDS is giving the protein 559 00:38:18,360 --> 00:38:20,520 a large net negative charge. 560 00:38:20,520 --> 00:38:22,980 So it's going to override whatever the intrinsic charge 561 00:38:22,980 --> 00:38:25,410 is of the protein. 562 00:38:25,410 --> 00:38:30,270 And so, it gives all proteins a similar mass-to-charge ratio 563 00:38:30,270 --> 00:38:31,500 here. 564 00:38:31,500 --> 00:38:33,000 With that said, sometimes, there are 565 00:38:33,000 --> 00:38:35,790 proteins that migrate in the gel in a manner that's 566 00:38:35,790 --> 00:38:38,040 not reflective of their molecular weight. 567 00:38:38,040 --> 00:38:40,390 That's just something to keep an eye out on. 568 00:38:40,390 --> 00:38:42,810 So within the slides that will be posted on Stellar, 569 00:38:42,810 --> 00:38:44,790 there'll be some background information 570 00:38:44,790 --> 00:38:46,490 about both of these methods-- 571 00:38:46,490 --> 00:38:49,380 the IEF gel and SDS-PAGE, which I 572 00:38:49,380 --> 00:38:53,330 encourage you to take a look there. 573 00:38:53,330 --> 00:38:57,570 OK, so back to the 2-D gel-- 574 00:38:57,570 --> 00:38:59,430 how is this actually going to be run? 575 00:39:08,660 --> 00:39:10,640 So it's one gel. 576 00:39:10,640 --> 00:39:16,760 First, it needs to run the IEF gel. 577 00:39:16,760 --> 00:39:18,530 And you need a special apparatus for. 578 00:39:18,530 --> 00:39:21,080 This it's called a cylinder, or tube, gel-- 579 00:39:21,080 --> 00:39:23,780 so not flat like what you're all accustomed to for SDS-PAGE. 580 00:39:31,010 --> 00:39:42,610 Then, this gel needs to be equilibrated in the SDS-PAGE 581 00:39:42,610 --> 00:39:43,110 buffer. 582 00:39:50,360 --> 00:39:52,880 And then, you run the SDS-PAGE separation. 583 00:40:02,120 --> 00:40:05,840 And, in this step, just to note, the gel is rotated 90 degrees. 584 00:40:16,480 --> 00:40:18,070 OK, so what you get-- 585 00:40:26,200 --> 00:40:30,330 you get a gel where we have molecular weight here. 586 00:40:30,330 --> 00:40:33,260 We have pI here. 587 00:40:33,260 --> 00:40:34,740 And if it's a cell lysate, there's 588 00:40:34,740 --> 00:40:37,700 going to be many, many spots. 589 00:40:37,700 --> 00:40:39,720 These should all be spots unless you 590 00:40:39,720 --> 00:40:41,600 did a poor job running the gel. 591 00:40:55,680 --> 00:40:58,260 So this 2-D gel is being used, because it's 592 00:40:58,260 --> 00:41:01,350 going to provide better separation than a standard 1-D 593 00:41:01,350 --> 00:41:02,040 gel. 594 00:41:02,040 --> 00:41:05,920 Imagine trying to separate peptides out of some cell 595 00:41:05,920 --> 00:41:09,330 lysate using just a 1-D gel. 596 00:41:09,330 --> 00:41:11,220 Even after this immunoprecipitation, 597 00:41:11,220 --> 00:41:17,400 we'll see that these samples are very complicated here for that. 598 00:41:17,400 --> 00:41:22,950 So what we need is some way to detect the spots that indicate 599 00:41:22,950 --> 00:41:24,450 different polypeptides. 600 00:41:24,450 --> 00:41:26,910 So what are methods? 601 00:41:26,910 --> 00:41:30,490 Maybe Coomassie stain for total protein. 602 00:41:30,490 --> 00:41:33,050 We can use the radiolabel-- 603 00:41:33,050 --> 00:41:36,780 autoradiography, for instance, which is what's done here. 604 00:41:36,780 --> 00:41:39,480 We're looking at the S35 radiolabel-- 605 00:41:39,480 --> 00:41:44,350 or maybe Western blot here. 606 00:41:44,350 --> 00:41:48,570 So how are we going to get from this gel 607 00:41:48,570 --> 00:41:51,620 to knowing the identity of each of these spots? 608 00:42:05,850 --> 00:42:07,820 AUDIENCE: You have to identify your spot, 609 00:42:07,820 --> 00:42:11,300 excise it, extract the protein from the gel, 610 00:42:11,300 --> 00:42:15,140 adjust it, and then run NS and line it up 611 00:42:15,140 --> 00:42:17,420 with known protein for evidence. 612 00:42:17,420 --> 00:42:18,930 ELIZABETH NOLAN: Exactly. 613 00:42:18,930 --> 00:42:23,780 So what will be done is that each spot of interest 614 00:42:23,780 --> 00:42:27,260 will be cut out of the gel. 615 00:42:27,260 --> 00:42:28,760 So you need a way to mark them. 616 00:42:28,760 --> 00:42:32,690 You'll see they're numbered in the data that we'll look at. 617 00:42:32,690 --> 00:42:37,220 The protein needs to be extracted out of the gel. 618 00:42:37,220 --> 00:42:39,920 Then the protein will be incubated 619 00:42:39,920 --> 00:42:45,530 with a protease that will give some number of fragments. 620 00:42:45,530 --> 00:42:47,870 Trypsin was used in this work. 621 00:42:47,870 --> 00:42:50,990 And then that digest can be analyzed by mass spec. 622 00:42:50,990 --> 00:42:54,920 And so, for each sample, you get all of the m over z values 623 00:42:54,920 --> 00:42:58,460 for the different polypeptides that resulted from the digest. 624 00:42:58,460 --> 00:43:00,740 And then, effectively, you can compare that 625 00:43:00,740 --> 00:43:04,490 to some database of E. coli protein sequences. 626 00:43:08,020 --> 00:43:12,960 So further details are provided throughout here. 627 00:43:12,960 --> 00:43:15,610 So what are the major questions? 628 00:43:15,610 --> 00:43:19,680 And what are we going to look for answers for in the data 629 00:43:19,680 --> 00:43:20,730 here? 630 00:43:20,730 --> 00:43:25,740 So first, how many proteins interact with GroEL? 631 00:43:25,740 --> 00:43:28,260 We can imagine getting an answer to this 632 00:43:28,260 --> 00:43:31,980 by counting the number of spots. 633 00:43:31,980 --> 00:43:34,590 What are the identities and structural features 634 00:43:34,590 --> 00:43:39,240 and properties of the proteins that interact with GroEL? 635 00:43:39,240 --> 00:43:43,530 We're going to get that from the mass spec analysis and then 636 00:43:43,530 --> 00:43:45,270 literature studies. 637 00:43:45,270 --> 00:43:49,260 And then another question we can get at is asking, 638 00:43:49,260 --> 00:43:51,820 how long do proteins interact with GroEL? 639 00:43:51,820 --> 00:43:54,870 Because we're calling the pulse-chase samples 640 00:43:54,870 --> 00:43:56,850 were taken at various time points 641 00:43:56,850 --> 00:43:59,050 over that 10-minute period. 642 00:43:59,050 --> 00:44:02,370 So, at two minutes, do we see the same polypeptides 643 00:44:02,370 --> 00:44:05,700 associated as we see at 10 minutes? 644 00:44:05,700 --> 00:44:08,880 Or if we monitor one given polypeptide, 645 00:44:08,880 --> 00:44:10,920 when does it show up and potentially 646 00:44:10,920 --> 00:44:13,530 disappear from the gels? 647 00:44:13,530 --> 00:44:18,090 So all of these samples can be addressed with these methods. 648 00:44:18,090 --> 00:44:21,870 And where we'll begin on Friday is 649 00:44:21,870 --> 00:44:24,820 going through the data in some detail. 650 00:44:24,820 --> 00:44:27,700 But just as a prelude to that in the last minute, 651 00:44:27,700 --> 00:44:30,435 here's the data from the paper for these gels. 652 00:44:33,960 --> 00:44:40,440 So this is looking at the 2-D gels for, on the top, 653 00:44:40,440 --> 00:44:44,430 total soluble cytoplasmic proteins at zero minutes 654 00:44:44,430 --> 00:44:47,220 and then total cytoplasmic proteins at 10 minutes. 655 00:44:47,220 --> 00:44:50,170 So this is without the immunoprecipitation. 656 00:44:50,170 --> 00:44:53,250 And then, at the bottom here, what we're looking at 657 00:44:53,250 --> 00:44:55,980 are the polypeptides that we're isolated 658 00:44:55,980 --> 00:45:00,840 from the immunoprecipitation with the anti-GroEL antibody 659 00:45:00,840 --> 00:45:03,360 at zero minutes and 10 minutes. 660 00:45:03,360 --> 00:45:05,830 And so, before we meet next time, 661 00:45:05,830 --> 00:45:09,810 what I encourage you to do is take a close look at these gels 662 00:45:09,810 --> 00:45:13,260 and see what information can you pull out just 663 00:45:13,260 --> 00:45:15,480 from a qualitative look. 664 00:45:15,480 --> 00:45:20,983 So simple questions, like, we see a lot of proteins here. 665 00:45:20,983 --> 00:45:23,150 And please don't go and try and count all the spots. 666 00:45:23,150 --> 00:45:25,290 I'll give you the numbers next time. 667 00:45:28,140 --> 00:45:30,870 How do these gels here from the immunoprecipitation 668 00:45:30,870 --> 00:45:33,090 differ from these up top? 669 00:45:33,090 --> 00:45:35,220 And it's not just the total number of proteins. 670 00:45:35,220 --> 00:45:37,880 There's some additional subtleties in these data. 671 00:45:37,880 --> 00:45:41,370 OK, so next time we'll begin examining these data, 672 00:45:41,370 --> 00:45:45,150 looking at what polypeptides were pulled down. 673 00:45:45,150 --> 00:45:50,010 And then we'll move into looking at the chaperone DnaK, 674 00:45:50,010 --> 00:45:53,360 DnaKJ system there.