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 --> 00:00:18,540 at ocw.mit.edu. 8 00:00:25,780 --> 00:00:29,600 ELIZABETH NOLAN: So where we're going to begin today 9 00:00:29,600 --> 00:00:32,390 is continuing with our discussions 10 00:00:32,390 --> 00:00:40,010 of the substrates for groEL, groES, and analysis 11 00:00:40,010 --> 00:00:42,350 of the data. 12 00:00:42,350 --> 00:00:49,710 And after that we'll talk about the DNAK DNAJ chaperone system 13 00:00:49,710 --> 00:00:50,210 here. 14 00:00:53,490 --> 00:00:58,520 So recall last time we left off with the question 15 00:00:58,520 --> 00:01:01,570 of the groEL groES substrate. 16 00:01:01,570 --> 00:01:05,090 So inside an E coli cell, what are the polypeptides 17 00:01:05,090 --> 00:01:08,960 that are folded by this macromolecular machine? 18 00:01:08,960 --> 00:01:18,120 And so there was the pulse chase experiment, 19 00:01:18,120 --> 00:01:32,430 there was immuno precipitation, and then analysis. 20 00:01:32,430 --> 00:01:34,260 And so in this analysis, we talked 21 00:01:34,260 --> 00:01:40,680 about doing two dimensional gel electrophoresis, and then 22 00:01:40,680 --> 00:01:45,090 trypsin digest and mass spec of the various spots. 23 00:01:45,090 --> 00:01:48,150 So where we left off were with these data 24 00:01:48,150 --> 00:01:53,070 here and the question, how many polypeptide substrates 25 00:01:53,070 --> 00:01:58,800 interact with groEL in vivo, so inside an E coli cell? 26 00:01:58,800 --> 00:02:04,590 And what we're looking at are the various gels 27 00:02:04,590 --> 00:02:07,830 for either total soluble cytoplasmic proteins 28 00:02:07,830 --> 00:02:10,690 on top at either 0 minutes-- 29 00:02:10,690 --> 00:02:12,300 so at the start of the pulse-- 30 00:02:12,300 --> 00:02:15,000 recall that these cells were treated with radio 31 00:02:15,000 --> 00:02:16,920 labeled methionine, and then there 32 00:02:16,920 --> 00:02:19,140 was a chase for a period of time when 33 00:02:19,140 --> 00:02:22,000 excess unlabeled methionine was added. 34 00:02:22,000 --> 00:02:25,050 So here we're looking at total soluble cytoplasm proteins 35 00:02:25,050 --> 00:02:27,630 10 minutes into the chase. 36 00:02:27,630 --> 00:02:29,130 And then at the bottom, what we're 37 00:02:29,130 --> 00:02:33,840 looking at are the polypeptides that were immunoprecipitated 38 00:02:33,840 --> 00:02:36,240 by treatment of this cell lisate say 39 00:02:36,240 --> 00:02:38,850 with the anti groEL antibody. 40 00:02:38,850 --> 00:02:42,150 So the idea is this antibody will bind to groEL, 41 00:02:42,150 --> 00:02:44,490 and if polypeptides are bound those 42 00:02:44,490 --> 00:02:46,397 will be pulled down as well. 43 00:02:46,397 --> 00:02:48,480 So it's kind of incredible this experiment worked. 44 00:02:48,480 --> 00:02:50,520 There was a bunch of questions after class 45 00:02:50,520 --> 00:02:54,840 in terms of the details of this immunoprecipitation just 46 00:02:54,840 --> 00:02:57,360 to think about is it a groEL monomer, 47 00:02:57,360 --> 00:02:59,430 or is it a groEL heptamer? 48 00:02:59,430 --> 00:03:01,920 How tightly are these polypeptides bound? 49 00:03:01,920 --> 00:03:05,590 How do they stay bound during the course of the workup? 50 00:03:05,590 --> 00:03:07,440 Where's groES? 51 00:03:07,440 --> 00:03:10,170 These are a number of questions to think about 52 00:03:10,170 --> 00:03:14,520 and to look at the experimental to see about answers. 53 00:03:14,520 --> 00:03:17,280 So where we're going to focus is right now 54 00:03:17,280 --> 00:03:18,600 looking at these gels. 55 00:03:18,600 --> 00:03:21,450 And so what we need to ask is, what do we learn just 56 00:03:21,450 --> 00:03:25,530 from qualitative inspection of these data? 57 00:03:25,530 --> 00:03:31,170 So on these along the y-axis we have molecular weight, 58 00:03:31,170 --> 00:03:34,530 and along the x-axis the PI. 59 00:03:34,530 --> 00:03:38,040 So if we first take a look at the total soluble cytoplasmic 60 00:03:38,040 --> 00:03:42,180 proteins at zero minutes and 10 minutes, what do we see? 61 00:03:50,880 --> 00:03:54,530 Do we see many spots or a few spots? 62 00:03:54,530 --> 00:03:56,550 Many spots, right? 63 00:03:56,550 --> 00:04:00,840 And we see many spots both at 0 minutes and at 10 minutes. 64 00:04:00,840 --> 00:04:04,980 So the E coli genome encodes over 4,000 proteins-- 65 00:04:04,980 --> 00:04:07,350 roughly 4,300. 66 00:04:07,350 --> 00:04:12,660 And if one were to go and count all of these spots, how many do 67 00:04:12,660 --> 00:04:13,350 we see? 68 00:04:13,350 --> 00:04:16,230 It's on the order of 2,500. 69 00:04:16,230 --> 00:04:18,720 So they detected on the order of 2,500 70 00:04:18,720 --> 00:04:24,660 different cytoplasmic proteins on these gels. 71 00:04:24,660 --> 00:04:26,970 What do we see in terms of distribution 72 00:04:26,970 --> 00:04:28,080 by molecular weight? 73 00:04:30,780 --> 00:04:34,110 Is it a broad distribution, or narrow distribution? 74 00:04:37,460 --> 00:04:41,050 Broad, we're seeing spots of all different molecular weights, 75 00:04:41,050 --> 00:04:44,250 so from low to high on this gel. 76 00:04:44,250 --> 00:04:46,654 What about PI? 77 00:04:46,654 --> 00:04:47,737 AUDIENCE: It's also broad. 78 00:04:47,737 --> 00:04:49,821 ELIZABETH NOLAN: We also have a broad distribution 79 00:04:49,821 --> 00:04:50,700 in these gels, right? 80 00:04:50,700 --> 00:04:54,150 So we see polypeptides of low through high PI 81 00:04:54,150 --> 00:04:57,420 on this scale from 4 to 7. 82 00:04:57,420 --> 00:05:00,840 So now what we want to do is look at the gels obtained 83 00:05:00,840 --> 00:05:04,990 for the samples from the immunoprecipitation and ask 84 00:05:04,990 --> 00:05:08,070 what do we see, and is that the same or different from what 85 00:05:08,070 --> 00:05:12,630 we see for the total cytoplasmic proteins up here? 86 00:05:12,630 --> 00:05:17,280 So if we look at the data here which are the polypeptides that 87 00:05:17,280 --> 00:05:20,280 were obtained from immunoprecipitation at 0 88 00:05:20,280 --> 00:05:22,140 minutes, what do we see? 89 00:05:27,290 --> 00:05:29,855 So do we see a few spots, a lot of spots? 90 00:05:33,581 --> 00:05:37,445 AUDIENCE: It's still a lot, and it's still distributed 91 00:05:37,445 --> 00:05:40,267 over a pretty wide range. 92 00:05:40,267 --> 00:05:42,600 ELIZABETH NOLAN: OK, so let's start with the first point 93 00:05:42,600 --> 00:05:45,810 Kenny made, which is that we have a lot of spots, 94 00:05:45,810 --> 00:05:47,490 and I'd argue that's true. 95 00:05:47,490 --> 00:05:51,510 In this gel, we see many spots where each spot indicates 96 00:05:51,510 --> 00:05:53,580 a distinct polypeptide. 97 00:05:53,580 --> 00:05:56,880 Do we see the same or less than here 98 00:05:56,880 --> 00:05:59,364 for the total cytoplasmic protein? 99 00:05:59,364 --> 00:06:00,490 AUDIENCE: It's less. 100 00:06:00,490 --> 00:06:01,990 ELIZABETH NOLAN: We see less, right? 101 00:06:01,990 --> 00:06:03,810 AUDIENCE: And they seem more concentrated. 102 00:06:03,810 --> 00:06:06,670 ELIZABETH NOLAN: Yeah, just wait a second. 103 00:06:06,670 --> 00:06:10,170 Right, so we see less, and that's a good sign 104 00:06:10,170 --> 00:06:12,450 because an antibody was used to pull down 105 00:06:12,450 --> 00:06:15,480 some fraction of this pool. 106 00:06:15,480 --> 00:06:17,430 So about how many are here? 107 00:06:17,430 --> 00:06:22,350 They found about 250 to 300 polypeptides there, 108 00:06:22,350 --> 00:06:25,170 so about 10% of these cytoplasmic proteins 109 00:06:25,170 --> 00:06:27,750 were found to be interacting here. 110 00:06:27,750 --> 00:06:30,450 So on the basis of the experiment, 111 00:06:30,450 --> 00:06:33,600 we can conclude these are polypeptides 112 00:06:33,600 --> 00:06:36,480 that interact with groEL here. 113 00:06:36,480 --> 00:06:39,270 OK so now Kenny has a few additional observations 114 00:06:39,270 --> 00:06:39,930 in this gel. 115 00:06:39,930 --> 00:06:41,050 What are those? 116 00:06:41,050 --> 00:06:44,410 So how are these polypeptides distributed? 117 00:06:44,410 --> 00:06:46,320 And we'll just focus on C for the moment. 118 00:06:48,880 --> 00:06:53,432 So in terms of molecular weight, what do we see? 119 00:06:53,432 --> 00:06:56,360 AUDIENCE: It's all scattered pretty wide range 120 00:06:56,360 --> 00:06:57,824 of molecular weights. 121 00:07:00,695 --> 00:07:02,570 ELIZABETH NOLAN: And so we have a wide range, 122 00:07:02,570 --> 00:07:04,820 and where is that range and how does 123 00:07:04,820 --> 00:07:07,310 that range compare to here? 124 00:07:07,310 --> 00:07:10,880 So I agree, but look at the subtleties. 125 00:07:10,880 --> 00:07:15,440 AUDIENCE: Most of them are above 8 kilodaltons? 126 00:07:15,440 --> 00:07:19,260 ELIZABETH NOLAN: Yeah, so let's roughly say in the range of 20. 127 00:07:19,260 --> 00:07:22,800 So if we look at the bottom part of the gel versus the top part 128 00:07:22,800 --> 00:07:25,440 of the gel here, and we compare that 129 00:07:25,440 --> 00:07:27,480 to the bottom part of the gel here 130 00:07:27,480 --> 00:07:29,700 and the top part of the gel here, 131 00:07:29,700 --> 00:07:31,650 we see some differences that aren't just 132 00:07:31,650 --> 00:07:34,760 the total number of spots. 133 00:07:34,760 --> 00:07:35,260 Rebecca? 134 00:07:35,260 --> 00:07:37,410 AUDIENCE: So it's like the ones that are smaller-- 135 00:07:37,410 --> 00:07:40,630 so the spots that respond to the smaller proteins, 136 00:07:40,630 --> 00:07:43,380 they seem to be more highly charged. 137 00:07:43,380 --> 00:07:45,080 ELIZABETH NOLAN: More highly charged. 138 00:07:45,080 --> 00:07:47,850 Yeah, so let's first stick to the size. 139 00:07:47,850 --> 00:07:52,440 So we're seeing that in the bottom region of this gel where 140 00:07:52,440 --> 00:07:54,750 we have lower molecular weight species, 141 00:07:54,750 --> 00:07:59,800 we see fewer of these here than here. 142 00:07:59,800 --> 00:08:01,980 So why might that be if there's less 143 00:08:01,980 --> 00:08:04,620 polypeptides with molecular weight 144 00:08:04,620 --> 00:08:06,480 smaller than 20 kilodaltons? 145 00:08:10,720 --> 00:08:11,920 Steve? 146 00:08:11,920 --> 00:08:14,560 AUDIENCE: If you just consider the total number 147 00:08:14,560 --> 00:08:16,350 of possible confirmations of protein 148 00:08:16,350 --> 00:08:19,960 can adopt or peptide to adopt as a exponential function 149 00:08:19,960 --> 00:08:23,050 of its size, larger proteins are more 150 00:08:23,050 --> 00:08:25,630 likely to have more non-productive 151 00:08:25,630 --> 00:08:27,472 folding pathways. 152 00:08:27,472 --> 00:08:29,680 So it's just less likely to have something that needs 153 00:08:29,680 --> 00:08:31,020 a chaperone at a smaller size. 154 00:08:31,020 --> 00:08:33,520 ELIZABETH NOLAN: Right, so maybe these smaller polypeptides, 155 00:08:33,520 --> 00:08:34,720 they need less help. 156 00:08:34,720 --> 00:08:37,150 Their domain structure is more simple. 157 00:08:37,150 --> 00:08:39,789 For instance, they're easier to fold, 158 00:08:39,789 --> 00:08:44,169 and other machinery can take care of that here. 159 00:08:44,169 --> 00:08:47,620 And then if we look at PI, what do we see? 160 00:08:47,620 --> 00:08:51,550 So how is the distribution in terms of PI? 161 00:08:55,652 --> 00:08:58,110 AUDIENCE: Large molecular weight proteins are pretty evenly 162 00:08:58,110 --> 00:09:02,953 distributed, but the smaller ones have more of a charge. 163 00:09:02,953 --> 00:09:03,870 ELIZABETH NOLAN: Yeah. 164 00:09:03,870 --> 00:09:07,260 How do you use the word charged? 165 00:09:07,260 --> 00:09:09,720 AUDIENCE: Sorry, I was looking at the scale. 166 00:09:09,720 --> 00:09:13,250 They actually have a PI closer to 7. 167 00:09:13,250 --> 00:09:16,410 ELIZABETH NOLAN: Yeah, just like you heard in recitations 2 168 00:09:16,410 --> 00:09:20,520 and 3, pay attention to the scale and what kind of charge-- 169 00:09:20,520 --> 00:09:22,020 if you're talking about charge, you 170 00:09:22,020 --> 00:09:24,990 have negatively charged and positively charged amino acids. 171 00:09:24,990 --> 00:09:27,840 So where in that regime are you? 172 00:09:27,840 --> 00:09:30,510 But if we look at these areas here, 173 00:09:30,510 --> 00:09:32,910 we see a wide distribution. 174 00:09:32,910 --> 00:09:34,470 And maybe when they're smaller we're 175 00:09:34,470 --> 00:09:36,960 seeing some more over here, but then ask yourself, 176 00:09:36,960 --> 00:09:41,550 is 22 an outlier there? 177 00:09:41,550 --> 00:09:48,510 So what can be done in terms of these data? 178 00:09:48,510 --> 00:09:52,620 This is actually an analysis of the gels looking 179 00:09:52,620 --> 00:09:56,010 at total proteins and groEL bound proteins 180 00:09:56,010 --> 00:09:59,550 for the total percentage in terms of PI 181 00:09:59,550 --> 00:10:01,540 and in terms of molecular weight. 182 00:10:01,540 --> 00:10:04,410 And so you can compare. 183 00:10:04,410 --> 00:10:10,440 And so what we see is that overall, and look a bit closer, 184 00:10:10,440 --> 00:10:13,410 that PI distributions are quite similar. 185 00:10:13,410 --> 00:10:16,260 Molecular weight we see some differences. 186 00:10:16,260 --> 00:10:18,330 We also don't see that many proteins 187 00:10:18,330 --> 00:10:20,460 that are greater than 90 kilodaltons being 188 00:10:20,460 --> 00:10:22,980 folded by this machine. 189 00:10:22,980 --> 00:10:25,650 And then again, why might that be? 190 00:10:25,650 --> 00:10:28,560 We learn that the chamber can accommodate polypeptides up 191 00:10:28,560 --> 00:10:31,050 to about 60 kilodaltons, so maybe they're 192 00:10:31,050 --> 00:10:33,960 just too big here. 193 00:10:33,960 --> 00:10:40,170 So what are the identities of these proteins here? 194 00:10:40,170 --> 00:10:42,570 So this is where the trypsin digest and mass 195 00:10:42,570 --> 00:10:44,950 spec comes into play. 196 00:10:44,950 --> 00:10:48,000 So you can imagine extracting the spots, 197 00:10:48,000 --> 00:10:50,040 digesting them with the protease trypsin, 198 00:10:50,040 --> 00:10:52,590 and then doing mass spec analysis to find out 199 00:10:52,590 --> 00:10:56,940 the identities and comparing that data to databases 200 00:10:56,940 --> 00:10:58,830 of E coli proteins. 201 00:10:58,830 --> 00:11:03,510 And so from that, of the 250 to 300 proteins that they 202 00:11:03,510 --> 00:11:06,510 identified in these immuno precipitation gels, 203 00:11:06,510 --> 00:11:11,040 they were able to identify 52 without a doubt. 204 00:11:11,040 --> 00:11:13,920 And what are some of those 52 proteins? 205 00:11:13,920 --> 00:11:17,730 So I've just highlighted a few examples. 206 00:11:17,730 --> 00:11:19,920 What do we see? 207 00:11:19,920 --> 00:11:24,750 So here's our friend DFTU as one example. 208 00:11:24,750 --> 00:11:29,050 We see subunit of RNA polymerase, ferritin, 209 00:11:29,050 --> 00:11:31,920 and certain rhibosomal proteins. 210 00:11:31,920 --> 00:11:37,230 So just thinking about these proteins and their role 211 00:11:37,230 --> 00:11:42,420 in translation, in RNA polarization, 212 00:11:42,420 --> 00:11:45,340 ferritin is an iron storage protein. 213 00:11:45,340 --> 00:11:47,768 What do we think? 214 00:11:47,768 --> 00:11:49,560 What are our thoughts about these proteins? 215 00:11:59,560 --> 00:12:02,860 They're pretty important, right? 216 00:12:02,860 --> 00:12:06,550 Imagine if EFTU you couldn't adopt its native confirmation. 217 00:12:06,550 --> 00:12:09,760 There might be some major problems. 218 00:12:09,760 --> 00:12:12,490 And recall when I introduced groEL, 219 00:12:12,490 --> 00:12:16,420 groES, we learned that they fall into the category 220 00:12:16,420 --> 00:12:20,380 of chaperonin, so they're essential for life. 221 00:12:20,380 --> 00:12:24,070 So that makes sense in terms of seeing some of these proteins 222 00:12:24,070 --> 00:12:26,560 as being very important. 223 00:12:26,560 --> 00:12:28,330 And what about structural motifs? 224 00:12:28,330 --> 00:12:32,110 It's then we see, OK, these are the 50 proteins we identified, 225 00:12:32,110 --> 00:12:33,700 what are their structural features, 226 00:12:33,700 --> 00:12:37,030 and what does that tell us about this chaperone? 227 00:12:37,030 --> 00:12:40,030 The conclusion is that overall, the proteins 228 00:12:40,030 --> 00:12:44,440 identified have quite complex structural features. 229 00:12:44,440 --> 00:12:50,260 So these can range from complex domain organization 230 00:12:50,260 --> 00:12:53,230 to beta sheets, including those that are buried 231 00:12:53,230 --> 00:12:57,220 and have large hydrophobic surfaces here. 232 00:12:57,220 --> 00:13:01,090 And so we can speculate that maybe some 233 00:13:01,090 --> 00:13:04,540 of these hydrophobic surfaces interact 234 00:13:04,540 --> 00:13:07,930 with the groEL-applicable domain to have these polypeptides 235 00:13:07,930 --> 00:13:09,625 enter into the chamber. 236 00:13:09,625 --> 00:13:10,750 Here, was there a question? 237 00:13:13,450 --> 00:13:16,370 AUDIENCE: Well, I was going to ask, I don't know for ferritin, 238 00:13:16,370 --> 00:13:18,820 but I know that you need a lot of ferritin molecules 239 00:13:18,820 --> 00:13:21,790 to form the thing. 240 00:13:21,790 --> 00:13:23,407 But all of those are also-- 241 00:13:23,407 --> 00:13:25,240 and again it's only 4 out of 52, but they're 242 00:13:25,240 --> 00:13:27,780 all proteins that exist in relatively high abundances. 243 00:13:27,780 --> 00:13:29,950 So could you also be making the argument 244 00:13:29,950 --> 00:13:32,493 that proteins that are more likely to have 245 00:13:32,493 --> 00:13:33,910 high concentrations, and therefore 246 00:13:33,910 --> 00:13:36,610 a higher probability of aggregating 247 00:13:36,610 --> 00:13:39,950 just because it's a prime molecular reaction 248 00:13:39,950 --> 00:13:46,283 could favor binding to groEL? 249 00:13:46,283 --> 00:13:48,200 ELIZABETH NOLAN: Yeah, I even thought about it 250 00:13:48,200 --> 00:13:51,575 in terms of they certainly are abundant. 251 00:13:55,880 --> 00:13:57,883 It could be, I just don't know. 252 00:13:57,883 --> 00:14:00,410 AUDIENCE: The experimental setup also biased it 253 00:14:00,410 --> 00:14:02,150 towards more abundant proteins. 254 00:14:02,150 --> 00:14:03,650 ELIZABETH NOLAN: Yeah, so could that 255 00:14:03,650 --> 00:14:07,310 have happened in the experimental setup? 256 00:14:07,310 --> 00:14:09,080 It's a possibility. 257 00:14:09,080 --> 00:14:12,050 So we learned that what EFT was about 10% 258 00:14:12,050 --> 00:14:14,300 of all rhibosomal proteins. 259 00:14:14,300 --> 00:14:16,940 So that's something also to keep in mind, 260 00:14:16,940 --> 00:14:19,490 and a good thought there. 261 00:14:19,490 --> 00:14:21,860 So what else can we learn? 262 00:14:21,860 --> 00:14:24,710 One more observation from these experiments 263 00:14:24,710 --> 00:14:29,330 before we move on to DNA K J. So recall last time 264 00:14:29,330 --> 00:14:33,200 when we talked about the actual pulse chase experiment, 265 00:14:33,200 --> 00:14:37,160 they took samples at multiple time points. 266 00:14:37,160 --> 00:14:39,290 And so why did they do that? 267 00:14:39,290 --> 00:14:41,210 You can imagine doing this analysis 268 00:14:41,210 --> 00:14:43,580 not just at 0 minutes and 10 minutes, 269 00:14:43,580 --> 00:14:46,730 but at a variety of time points and ask, 270 00:14:46,730 --> 00:14:51,290 if we compare gel to gel and we compare spot to spot-- 271 00:14:51,290 --> 00:14:56,840 so going back, these spots are labeled many of them in here-- 272 00:14:56,840 --> 00:14:58,910 we can ask the question, how does the intensity 273 00:14:58,910 --> 00:15:01,070 of that spot change over time? 274 00:15:01,070 --> 00:15:03,500 And what does that tell us about the interactions 275 00:15:03,500 --> 00:15:09,410 of that polypeptide with groEL? 276 00:15:09,410 --> 00:15:24,640 So just for example, here. 277 00:15:24,640 --> 00:15:27,450 Example, just imagine at time equals 278 00:15:27,450 --> 00:15:40,194 0 we see some protein or polypeptide x. 279 00:15:40,194 --> 00:15:45,235 So then what happens at, say, time equals 2 minutes? 280 00:15:48,930 --> 00:15:53,480 If we do not see it, let's consider two options. 281 00:15:53,480 --> 00:16:12,570 Do not see x, maybe we conclude that x dissociates quickly 282 00:16:12,570 --> 00:16:15,070 or folds relatively easily. 283 00:16:19,270 --> 00:16:28,110 Imagine if we do see x after 2 minutes here, 284 00:16:28,110 --> 00:16:36,720 maybe the conclusion is x is not yet folded here. 285 00:16:40,770 --> 00:16:46,200 And then we can imagine doing this at different time points, 286 00:16:46,200 --> 00:16:49,320 and they went out to 10 minutes here. 287 00:16:49,320 --> 00:16:53,520 So maybe if we see x at 10 minutes, 288 00:16:53,520 --> 00:16:55,890 the conclusion is x is difficult to fold. 289 00:17:06,500 --> 00:17:09,230 And too, we want to think about these time points 290 00:17:09,230 --> 00:17:10,970 also from the standpoint of what we 291 00:17:10,970 --> 00:17:14,089 saw in terms of the residency time of a polypeptide 292 00:17:14,089 --> 00:17:16,640 in the groEL chamber. 293 00:17:16,640 --> 00:17:18,859 So we saw from the various models 294 00:17:18,859 --> 00:17:23,060 that that's somewhere on the order of 6 to 10 seconds. 295 00:17:23,060 --> 00:17:28,280 So there can be multiple binding and release events that occur. 296 00:17:28,280 --> 00:17:34,970 So in this paper, what the authors did is trace the spots 297 00:17:34,970 --> 00:17:39,410 and compare the intensities of the spots over time. 298 00:17:39,410 --> 00:17:43,160 And you can do a little exercise from these gels looking 299 00:17:43,160 --> 00:17:46,872 at spots they circled and just ask qualitatively, 300 00:17:46,872 --> 00:17:48,080 what's happening to the spot? 301 00:17:48,080 --> 00:17:50,240 Is the intensity staying the same? 302 00:17:50,240 --> 00:17:51,990 Is it being reduced? 303 00:17:51,990 --> 00:17:54,260 So for instance, it's easy to look at spot number 304 00:17:54,260 --> 00:18:01,160 22 here at 0 minutes versus spot 22 at 10 minutes. 305 00:18:01,160 --> 00:18:02,150 And what do we see? 306 00:18:09,280 --> 00:18:14,110 Does it look the same, more intense, less intense? 307 00:18:14,110 --> 00:18:15,730 Less intense, right? 308 00:18:15,730 --> 00:18:21,460 What about spot number 12 at 0 minutes versus 10 minutes? 309 00:18:24,850 --> 00:18:27,580 They look quite similar by eye. 310 00:18:27,580 --> 00:18:29,680 So you can imagine doing this type of exercise 311 00:18:29,680 --> 00:18:32,170 through each gel and actually doing it quantitatively 312 00:18:32,170 --> 00:18:34,570 using some instrumentation. 313 00:18:34,570 --> 00:18:37,750 So what do they see? 314 00:18:37,750 --> 00:18:41,710 Effectively in this, they divided the data 315 00:18:41,710 --> 00:18:45,880 into three groups based on certain trends. 316 00:18:45,880 --> 00:18:49,750 And that's shown here where what we're looking at 317 00:18:49,750 --> 00:18:53,830 is the relative intensity change versus time. 318 00:18:53,830 --> 00:18:55,630 So you can imagine at some time point 319 00:18:55,630 --> 00:18:58,180 that spot has a maximum intensity that they've 320 00:18:58,180 --> 00:19:00,580 put at 100. 321 00:19:00,580 --> 00:19:02,930 So we see the three groups here. 322 00:19:02,930 --> 00:19:05,140 And the question is if we look at these as groups, 323 00:19:05,140 --> 00:19:08,590 what do the data show? 324 00:19:08,590 --> 00:19:15,370 So in group one, we see examples where the spot at time equals 0 325 00:19:15,370 --> 00:19:18,330 is at a maximum, and then the intensity of that, 326 00:19:18,330 --> 00:19:20,960 so spots decrease over time. 327 00:19:20,960 --> 00:19:24,070 And the other thing we see is that at some time that 328 00:19:24,070 --> 00:19:28,240 isn't very long, the intensities go to approximately 0. 329 00:19:28,240 --> 00:19:32,230 So we're not seeing these polypeptides bound any longer. 330 00:19:32,230 --> 00:19:35,890 And then effectively what we want 331 00:19:35,890 --> 00:19:41,740 to ask is do these polypeptides have any similar features? 332 00:19:41,740 --> 00:19:46,690 And what the authors observed is that the polypeptides falling 333 00:19:46,690 --> 00:19:50,320 into this group showing this behavior 334 00:19:50,320 --> 00:19:53,260 are smaller than 60 kilodaltons. 335 00:19:53,260 --> 00:19:56,740 And as shown here, they're seeing them completely released 336 00:19:56,740 --> 00:19:58,930 over the time course of this experiment, 337 00:19:58,930 --> 00:20:02,680 and in general within the first 2 minutes. 338 00:20:02,680 --> 00:20:04,330 So what does that correspond to? 339 00:20:04,330 --> 00:20:09,040 How they interpreted this was that these polypeptides are 340 00:20:09,040 --> 00:20:13,680 either binding groEL once or have several rounds of binding 341 00:20:13,680 --> 00:20:15,520 and ultimately reached their folded state 342 00:20:15,520 --> 00:20:19,250 in this relatively short time period. 343 00:20:19,250 --> 00:20:21,340 So how does group two differ? 344 00:20:21,340 --> 00:20:23,740 Looking at these data, what do we 345 00:20:23,740 --> 00:20:26,020 see in group two that's different from group one? 346 00:20:41,796 --> 00:20:46,050 [INAUDIBLE] Yeah, we're seeing the relative intensity 347 00:20:46,050 --> 00:20:47,910 never go all the way to 0. 348 00:20:47,910 --> 00:20:56,100 So here we've gone to 0, here we see 20% to 30% as the cutoff. 349 00:20:56,100 --> 00:20:58,410 So how are these data interpreted in this work, 350 00:20:58,410 --> 00:21:01,590 and what are the identities of these polypeptides? 351 00:21:01,590 --> 00:21:04,560 So similar to group one, these polypeptides 352 00:21:04,560 --> 00:21:08,160 are also all smaller than 60 kilodaltons. 353 00:21:08,160 --> 00:21:10,860 And how this behavior is interpreted 354 00:21:10,860 --> 00:21:12,720 is that even after 10 minutes, there's 355 00:21:12,720 --> 00:21:16,260 some fraction of these polypeptides that are still 356 00:21:16,260 --> 00:21:18,720 associated with groEL. 357 00:21:18,720 --> 00:21:22,100 So they haven't reached their native fold 358 00:21:22,100 --> 00:21:23,100 and are remaining bound. 359 00:21:25,990 --> 00:21:29,770 What's going on in this group here, group three? 360 00:21:29,770 --> 00:21:31,785 This behavior is very different. 361 00:21:39,060 --> 00:21:41,430 AUDIENCE: You see peak intensity is a little bit later 362 00:21:41,430 --> 00:21:43,766 than the rest of them, and they also 363 00:21:43,766 --> 00:21:46,580 don't go to 0 after 10 minutes. 364 00:21:46,580 --> 00:21:47,760 ELIZABETH NOLAN: Yes. 365 00:21:47,760 --> 00:21:52,620 So these proteins are interacting with groEL 366 00:21:52,620 --> 00:21:54,510 because they were pulled down, but it 367 00:21:54,510 --> 00:21:58,140 looks like they're interacting at later time points. 368 00:21:58,140 --> 00:22:01,440 So we see this growth in terms of increase 369 00:22:01,440 --> 00:22:04,710 in intensity over time, and then they go down. 370 00:22:04,710 --> 00:22:09,390 And here we see 40% or higher. 371 00:22:09,390 --> 00:22:13,140 So they are not readily dissociating, 372 00:22:13,140 --> 00:22:15,570 binding at longer time points. 373 00:22:15,570 --> 00:22:19,035 So one question here is are these dead end species? 374 00:22:23,640 --> 00:22:26,520 And within this work, the authors 375 00:22:26,520 --> 00:22:28,290 did some additional controls which 376 00:22:28,290 --> 00:22:31,650 there's some detail in the notes I'll post in lecture. 377 00:22:31,650 --> 00:22:33,450 But effectively asking, what happens 378 00:22:33,450 --> 00:22:36,720 if we add in groES, what happens if we add in ATP? 379 00:22:36,720 --> 00:22:39,280 Do we still see these species or not? 380 00:22:39,280 --> 00:22:44,460 And some of them were released under those conditions there. 381 00:22:44,460 --> 00:22:48,570 So in summary, what we see from this 382 00:22:48,570 --> 00:22:55,080 is a method to look at chaperone substrate selection 383 00:22:55,080 --> 00:22:57,480 in the context of a cell. 384 00:22:57,480 --> 00:23:02,430 We see that groEL folds proteins over a range of sizes, 385 00:23:02,430 --> 00:23:03,710 but not really the small ones. 386 00:23:03,710 --> 00:23:06,660 So under 20 kilodaltons not so much, 387 00:23:06,660 --> 00:23:09,390 and over 60 kilodaltons not so much here, 388 00:23:09,390 --> 00:23:12,030 and that these polypeptide substrates 389 00:23:12,030 --> 00:23:14,760 have complex native folds. 390 00:23:14,760 --> 00:23:19,050 So where we're going to close the chaperone unit 391 00:23:19,050 --> 00:23:23,790 is with looking at the machinery DNA K J. 392 00:23:23,790 --> 00:23:26,670 And so we'll introduce that system 393 00:23:26,670 --> 00:23:29,250 and then look at a similar series of experiments 394 00:23:29,250 --> 00:23:33,150 where the substrate scope for this chaperone system 395 00:23:33,150 --> 00:23:35,430 was evaluated. 396 00:23:35,430 --> 00:23:41,280 So if we go back to the overview from the start 397 00:23:41,280 --> 00:23:43,920 where all of these players were introduced, 398 00:23:43,920 --> 00:23:46,090 this is where we are now. 399 00:23:46,090 --> 00:23:51,360 So we're looking at DNA K and its co-chaperone DNA J. 400 00:23:51,360 --> 00:23:53,670 So these are downstream of trigger factor. 401 00:23:53,670 --> 00:23:55,800 What do we have for DNA K and J? 402 00:24:04,120 --> 00:24:06,470 So these are heat shock proteins. 403 00:24:06,470 --> 00:24:09,100 DNA K is an HSP 70. 404 00:24:09,100 --> 00:24:14,390 So 70 kilodaltons, and HSP 70s are ubiquitous. 405 00:24:14,390 --> 00:24:16,240 So just to note, they're involved 406 00:24:16,240 --> 00:24:19,500 in a variety of protein quality control functions. 407 00:24:19,500 --> 00:24:21,310 So we have folding, as we'll talk 408 00:24:21,310 --> 00:24:25,540 about in the context of today's lecture in this module, 409 00:24:25,540 --> 00:24:29,740 but even rolls that range from protein transport to assisting 410 00:24:29,740 --> 00:24:32,830 with protein degradation occur. 411 00:24:32,830 --> 00:24:44,440 So here we have HSP 70 for DNA K and HSP 40 for DNA J. 412 00:24:44,440 --> 00:24:48,840 So in this system, DNA K is the chaperone 413 00:24:48,840 --> 00:24:54,950 and DNA J is the co-chaperone, and DNA K is ATP dependent. 414 00:24:54,950 --> 00:25:06,450 So it's monomeric. 415 00:25:06,450 --> 00:25:11,370 So with this system we don't have a chamber 416 00:25:11,370 --> 00:25:19,537 like we have with groEL, groES, and it's ATP dependent. 417 00:25:22,530 --> 00:25:33,460 DNA J is the co-chaperone here. 418 00:25:33,460 --> 00:25:37,780 So what happens in terms of this system? 419 00:25:37,780 --> 00:25:48,220 So effectively DNA J, the co-chaperone, 420 00:25:48,220 --> 00:26:08,130 scans hydrophobic surfaces of proteins or polypeptides, 421 00:26:08,130 --> 00:26:10,080 and it associates with them so it binds. 422 00:26:15,010 --> 00:26:20,610 And then what DNA J does is it delivers 423 00:26:20,610 --> 00:26:35,270 non-native polypeptides to DNA K. 424 00:26:35,270 --> 00:26:41,420 And then how we think about DNA K 425 00:26:41,420 --> 00:26:49,880 is that DNA K binds and releases unfolded polypeptides. 426 00:27:01,260 --> 00:27:02,880 And this is another case where there 427 00:27:02,880 --> 00:27:06,090 can be multiple cycles of binding and release. 428 00:27:06,090 --> 00:27:09,000 So DNA K will bind to a polypeptide that 429 00:27:09,000 --> 00:27:12,390 has an unfolded region, there'll be some period of time 430 00:27:12,390 --> 00:27:17,700 that that complex exists, and then DNA K will release it. 431 00:27:17,700 --> 00:27:20,700 And so in terms of where it likes to bind, 432 00:27:20,700 --> 00:27:23,970 these are typically six to nine amino acid 433 00:27:23,970 --> 00:27:29,450 segments that are hydrophobic. 434 00:27:36,830 --> 00:27:39,950 So it likes residues like leucine and isoleucine. 435 00:27:39,950 --> 00:27:43,130 And statistically, this type of region 436 00:27:43,130 --> 00:27:46,850 occurs about every 40 amino acids. 437 00:27:46,850 --> 00:27:50,630 And for these segments, just to note 438 00:27:50,630 --> 00:27:53,030 that there's a range of binding affinities. 439 00:27:53,030 --> 00:27:59,390 You can imagine there's a variety of possibilities here. 440 00:27:59,390 --> 00:28:02,090 And what's found from studies is that the KD 441 00:28:02,090 --> 00:28:05,180 of DNA K for various polypeptides 442 00:28:05,180 --> 00:28:15,420 can range from about 5 nanomolar to about 5 micromolar, 443 00:28:15,420 --> 00:28:17,760 so by several orders of magnitude. 444 00:28:20,760 --> 00:28:24,910 In terms of size of polypeptide, it 445 00:28:24,910 --> 00:28:28,440 stated that DNA K has some preference for polypeptides 446 00:28:28,440 --> 00:28:31,410 on the order of 20 to 30 kilodaltons, 447 00:28:31,410 --> 00:28:33,210 but it can bind larger ones and it 448 00:28:33,210 --> 00:28:36,720 can bind polypeptides greater than 60 kilodaltons, 449 00:28:36,720 --> 00:28:38,830 as we'll see later. 450 00:28:38,830 --> 00:28:41,490 So in this system there's another player 451 00:28:41,490 --> 00:28:53,340 that we need to think about, and that's this GrpE, or grip E. 452 00:28:53,340 --> 00:28:59,395 And what we have here is a nucleotide exchange factor. 453 00:29:06,210 --> 00:29:11,730 So any f, and it's also a thermal sensor. 454 00:29:17,230 --> 00:29:22,790 And what GrpE does is that it regulates 455 00:29:22,790 --> 00:29:37,570 DNA K binding to a substrate by inducing ADP release. 456 00:29:37,570 --> 00:29:41,350 So what we'll see is that the ATP and ADP bound 457 00:29:41,350 --> 00:29:44,140 forms of DNA K have different affinities 458 00:29:44,140 --> 00:29:47,760 for these polypeptide substrates. 459 00:29:47,760 --> 00:29:49,900 So what we're going to do is look 460 00:29:49,900 --> 00:29:54,610 at the structures of the components of this system 461 00:29:54,610 --> 00:29:56,890 and then look at the cycle. 462 00:29:56,890 --> 00:30:11,860 And so if we consider DNA K, so we think of this protein 463 00:30:11,860 --> 00:30:14,630 as having two different domains. 464 00:30:14,630 --> 00:30:18,280 So there's an N terminal domain and a C terminal domain. 465 00:30:29,590 --> 00:30:32,080 And in this end terminal domain what 466 00:30:32,080 --> 00:30:37,210 we have is the nucleotide binding domain, NBD. 467 00:30:37,210 --> 00:30:41,620 So this is where ATPase activity occurs, and this 468 00:30:41,620 --> 00:30:43,630 is about 44 kilodaltons here. 469 00:30:47,200 --> 00:30:54,270 There's a linker region, and then the C terminal, 470 00:30:54,270 --> 00:31:04,590 and we have the peptide binding or substrate binding domain. 471 00:31:04,590 --> 00:31:07,590 This is 27 kilodaltons. 472 00:31:10,140 --> 00:31:14,730 So here if we think about this part 473 00:31:14,730 --> 00:31:17,520 just in cartoon form, what's observed 474 00:31:17,520 --> 00:31:21,645 is that there's a cleft for binding ATP or ADP. 475 00:31:35,270 --> 00:31:43,220 So ATP or ADP binds here, and this 476 00:31:43,220 --> 00:31:45,950 is also where the nucleotide exchange factor 477 00:31:45,950 --> 00:31:48,170 GrpE will interact, because that's 478 00:31:48,170 --> 00:31:53,400 its job as a nucleotide exchange factor is to help with that. 479 00:31:53,400 --> 00:32:02,840 So basically we have GrpE here. 480 00:32:02,840 --> 00:32:07,910 What we see in this domain, it's often 481 00:32:07,910 --> 00:32:15,530 described as being a beta sandwich 482 00:32:15,530 --> 00:32:17,605 plus an alpha helical latch. 483 00:32:22,740 --> 00:32:34,950 And the idea is that this latch closes in the presence 484 00:32:34,950 --> 00:32:36,120 of the polypeptide. 485 00:32:45,600 --> 00:32:49,380 So effectively, if we look at this as a cartoon-- 486 00:32:49,380 --> 00:32:52,080 and we'll look at actual structures in a minute-- 487 00:32:52,080 --> 00:33:00,600 this peptide binding domain can either be in an open form, 488 00:33:00,600 --> 00:33:03,080 and this is the latch. 489 00:33:03,080 --> 00:33:06,600 You have the alpha helical part, here's the beta sandwich. 490 00:33:06,600 --> 00:33:11,310 And if there's some polypeptide to bind, what happens 491 00:33:11,310 --> 00:33:20,430 is that the latch closes and the polypeptide is bound here. 492 00:33:20,430 --> 00:33:27,120 So this is the closed form, and this pocket is hydrophobic. 493 00:33:33,820 --> 00:33:35,650 And that makes sense based on what 494 00:33:35,650 --> 00:33:41,290 we know about DNA K liking to bind hydrophobic stretches. 495 00:33:41,290 --> 00:33:46,780 So let's look at some structures of DNA K. I present 496 00:33:46,780 --> 00:33:51,040 two slides of structures here, one from the assigned review 497 00:33:51,040 --> 00:33:52,150 and this other version. 498 00:33:52,150 --> 00:33:56,050 And I'll just focus on this one for here. 499 00:33:56,050 --> 00:34:02,920 So here we're looking at the domain organization. 500 00:34:02,920 --> 00:34:08,239 What we have here is the nucleotide binding domain. 501 00:34:08,239 --> 00:34:11,679 So here's that clef for ATP binding. 502 00:34:11,679 --> 00:34:14,949 Here we're looking at the peptide binding domain. 503 00:34:14,949 --> 00:34:17,620 So the beta sandwich region is in green, 504 00:34:17,620 --> 00:34:20,679 the alpha helical latch is in yellow, 505 00:34:20,679 --> 00:34:23,590 and we see that there's a model polypeptide here, 506 00:34:23,590 --> 00:34:26,710 and this is in the closed form. 507 00:34:26,710 --> 00:34:31,969 Here's another view of DNA K with a peptide bound. 508 00:34:31,969 --> 00:34:37,420 So we see the beta sandwich, here's the alpha helical latch. 509 00:34:37,420 --> 00:34:40,239 This depiction here from the review 510 00:34:40,239 --> 00:34:45,489 is showing the closed and open states, and closed and open 511 00:34:45,489 --> 00:34:49,210 is referring to the green area here. 512 00:34:49,210 --> 00:34:53,170 So don't get confused with the nucleotide binding domain 513 00:34:53,170 --> 00:34:54,940 and how these are shown. 514 00:34:54,940 --> 00:34:56,860 So what we see here, again, there's 515 00:34:56,860 --> 00:35:00,980 a bound polypeptide in this peptide binding domain, 516 00:35:00,980 --> 00:35:03,700 and here there's no bound polypeptide. 517 00:35:03,700 --> 00:35:06,160 And we see that now this alpha helical region 518 00:35:06,160 --> 00:35:07,690 is sticking up there. 519 00:35:11,740 --> 00:35:13,280 So what about DNA J? 520 00:35:17,030 --> 00:35:32,250 You consider DNA J, we're just going 521 00:35:32,250 --> 00:35:41,650 to focus on the domain organization and just 522 00:35:41,650 --> 00:35:44,500 a more simplified view than what's on the slide. 523 00:35:47,500 --> 00:36:01,540 We have two domains for DNA K binding, 524 00:36:01,540 --> 00:36:04,360 and then for peptide binding. 525 00:36:09,930 --> 00:36:13,840 So DNA K is going to go out there and find some polypeptide 526 00:36:13,840 --> 00:36:16,630 that needs the help of DNA J. It's going to bind 527 00:36:16,630 --> 00:36:20,590 that polypeptide and deliver it to DNA K. So effectively, 528 00:36:20,590 --> 00:36:23,680 it interacts both with the polypeptide substrate 529 00:36:23,680 --> 00:36:29,620 and it also acts with DNA K when delivering this polypeptide. 530 00:36:29,620 --> 00:36:36,220 So just to point out DNA J is part of an HSP 40 family, 531 00:36:36,220 --> 00:36:39,310 and these are quite diverse. 532 00:36:39,310 --> 00:36:43,540 I just illustrate that from the range of different sizes, 533 00:36:43,540 --> 00:36:47,170 so from about 100 to about 2,000 amino acids. 534 00:36:47,170 --> 00:36:52,170 And all of these HSP 40s have what's called a J domain, 535 00:36:52,170 --> 00:36:54,580 and in this more detailed depiction here 536 00:36:54,580 --> 00:36:58,840 it's indicated these 70 amino acids at the N terminus 537 00:36:58,840 --> 00:37:01,570 are the J domain, and they're important for interacting 538 00:37:01,570 --> 00:37:04,760 with DNA K or another HSP 70. 539 00:37:07,670 --> 00:37:09,370 So what about GrpE? 540 00:37:18,140 --> 00:37:24,320 This nucleotide exchange factor, so GrpE is a homodimer. 541 00:37:30,900 --> 00:37:33,150 And if we just look at one monomer, 542 00:37:33,150 --> 00:37:34,740 and then I'll show you the structure. 543 00:37:39,000 --> 00:37:45,870 So in '97 a crystal structure of GrpE with DNA K nucleotide 544 00:37:45,870 --> 00:37:47,640 binding domain was published, and this 545 00:37:47,640 --> 00:37:49,320 is what came from that. 546 00:37:49,320 --> 00:37:52,320 So just use your imagination, maybe I'll draw this a little 547 00:37:52,320 --> 00:37:53,424 differently. 548 00:37:56,370 --> 00:38:08,790 Basically what we see with GrpE is that there's a beta sheet, 549 00:38:08,790 --> 00:38:10,720 and this is the C terminal region. 550 00:38:13,770 --> 00:38:18,420 And then what we see here is an extended alpha helix. 551 00:38:24,720 --> 00:38:26,430 And this is the end terminal region. 552 00:38:35,630 --> 00:38:40,480 And this is just a cartoon of the monomer. 553 00:38:40,480 --> 00:38:47,050 So what happens is that the GrpE homodimer uses 554 00:38:47,050 --> 00:38:50,320 one of the beta sheets of one monomer 555 00:38:50,320 --> 00:38:59,200 to insert into that ATP binding clef here of DNA K. 556 00:38:59,200 --> 00:39:03,550 And when that happens, it forces it open there. 557 00:39:03,550 --> 00:39:05,800 So let's look at the structure, and this 558 00:39:05,800 --> 00:39:08,800 is something that actually puzzled me for quite some time, 559 00:39:08,800 --> 00:39:11,150 but there's been a recent update. 560 00:39:11,150 --> 00:39:16,820 So this is a crystal structure of GrpE homodimer. 561 00:39:16,820 --> 00:39:21,640 So we see one monomer in blue and one monitoring green bound 562 00:39:21,640 --> 00:39:24,910 to an end terminal nucleotide binding 563 00:39:24,910 --> 00:39:29,170 domain of DNA K, which is shown in pink. 564 00:39:29,170 --> 00:39:33,070 And so we see the beta sheet region of each monomer, 565 00:39:33,070 --> 00:39:35,470 we see the extended alpha helix. 566 00:39:35,470 --> 00:39:39,550 The C terminal end is here, the N terminal end is here. 567 00:39:39,550 --> 00:39:42,700 And I note that not shown in this structure 568 00:39:42,700 --> 00:39:49,000 is there is an unfolded region after the end here of GrpE. 569 00:39:49,000 --> 00:39:53,860 And so we see this nucleotide binding domain interacting 570 00:39:53,860 --> 00:39:55,450 with one of the beta sheets. 571 00:39:55,450 --> 00:39:58,300 So a one to one stoichiometry. 572 00:39:58,300 --> 00:40:02,800 So the idea, as we'll see when we go forth with the cycle, 573 00:40:02,800 --> 00:40:07,000 is that GrpE is inserting the C terminal beta 574 00:40:07,000 --> 00:40:12,430 sheet into the nucleotide binding class of DNA K. 575 00:40:12,430 --> 00:40:15,160 And this happens for the ADP bound form, 576 00:40:15,160 --> 00:40:16,855 and it facilitates ADP release. 577 00:40:19,780 --> 00:40:23,680 So what's going on down here? 578 00:40:23,680 --> 00:40:27,550 Why is there this extended alpha helix? 579 00:40:27,550 --> 00:40:32,290 And I'll just note there is a study just in the past year 580 00:40:32,290 --> 00:40:35,920 where interactions between DNA K and GrpE 581 00:40:35,920 --> 00:40:38,360 were studied in some more detail. 582 00:40:38,360 --> 00:40:41,740 So they used some biochemical experiments, some cryo electron 583 00:40:41,740 --> 00:40:43,630 microscopy. 584 00:40:43,630 --> 00:40:46,930 And what they learned is that the interactions between GrpE 585 00:40:46,930 --> 00:40:50,410 and DNA K are more complex than what's seen here, 586 00:40:50,410 --> 00:40:52,650 and what they observe in their cryo 587 00:40:52,650 --> 00:40:58,990 EM is evidence for this N terminal region interacting 588 00:40:58,990 --> 00:41:02,260 with the substrate or polypeptide binding domain 589 00:41:02,260 --> 00:41:04,360 of DNA K. 590 00:41:04,360 --> 00:41:07,660 So there's some dynamics and flexibility 591 00:41:07,660 --> 00:41:10,840 that we can't appreciate from this crystal structure. 592 00:41:10,840 --> 00:41:15,700 And so that begs into question, how else is GrpE facilitating 593 00:41:15,700 --> 00:41:19,030 this cycle and modulating confirmation 594 00:41:19,030 --> 00:41:21,670 and function of DNA K? 595 00:41:21,670 --> 00:41:23,620 So you're not responsible for these details, 596 00:41:23,620 --> 00:41:25,370 but if it's something you're curious about 597 00:41:25,370 --> 00:41:29,740 I've included the reference. 598 00:41:29,740 --> 00:41:35,140 So effectively GrpE accelerates the release of ADP, 599 00:41:35,140 --> 00:41:40,040 and that in turn promotes binding of ATP. 600 00:41:40,040 --> 00:41:43,090 So what is the functional cycle? 601 00:41:43,090 --> 00:41:44,950 And we'll look at this depiction here. 602 00:41:44,950 --> 00:41:48,730 There's another depiction in the notes from the reading. 603 00:41:48,730 --> 00:41:50,600 This is the current model. 604 00:41:50,600 --> 00:41:54,070 And in this model, we're going to start here. 605 00:41:54,070 --> 00:41:56,170 So what do we see? 606 00:41:56,170 --> 00:42:00,730 We have DNA K in the ATP bound form. 607 00:42:00,730 --> 00:42:03,070 So we have the two domains-- 608 00:42:03,070 --> 00:42:06,970 the nucleotide binding domain, and here the polypeptide 609 00:42:06,970 --> 00:42:08,770 substrate binding domain. 610 00:42:08,770 --> 00:42:13,060 And in this cartoon, we see that alpha helical latch is open, 611 00:42:13,060 --> 00:42:15,910 so no polypeptides bound. 612 00:42:15,910 --> 00:42:19,690 And what we also see is that the ATPase activity here 613 00:42:19,690 --> 00:42:21,520 is very, very low. 614 00:42:21,520 --> 00:42:25,930 So DNA K is not hydrolyzing its ATP. 615 00:42:25,930 --> 00:42:27,390 So then what happens? 616 00:42:27,390 --> 00:42:31,150 DNA K-- sorry, DNA J, the co-chaperone, 617 00:42:31,150 --> 00:42:33,280 has found some polypeptide substrate-- 618 00:42:33,280 --> 00:42:35,170 indicated by this S-- 619 00:42:35,170 --> 00:42:38,830 that needs the help of DNA K. So J 620 00:42:38,830 --> 00:42:41,410 binds the polypeptide substrate, and it 621 00:42:41,410 --> 00:42:45,310 delivers that polypeptide to DNA K. 622 00:42:45,310 --> 00:42:46,940 So what does this cartoon tell us? 623 00:42:46,940 --> 00:42:49,840 It tells us that J is interacting with K, 624 00:42:49,840 --> 00:42:55,060 and here we see the polypeptide substrate being delivered. 625 00:42:55,060 --> 00:43:00,190 So when DNA K is in the ATP bound form, 626 00:43:00,190 --> 00:43:03,370 it binds peptides with relatively low affinity 627 00:43:03,370 --> 00:43:04,670 and in a reversible manner. 628 00:43:04,670 --> 00:43:07,150 So there's fast exchange, that polypeptide's 629 00:43:07,150 --> 00:43:09,220 going to come on and off. 630 00:43:09,220 --> 00:43:14,260 And when DNA J binds and delivers the polypeptide, 631 00:43:14,260 --> 00:43:19,060 it activates the ATPase activity of DNA K. 632 00:43:19,060 --> 00:43:21,310 So that's indicated here. 633 00:43:21,310 --> 00:43:26,240 So the ATPase activity is enhanced substantially 634 00:43:26,240 --> 00:43:30,010 so you can compare the values for some quantitative insight. 635 00:43:30,010 --> 00:43:32,350 There's ATP hydrolysis. 636 00:43:32,350 --> 00:43:37,770 ATP hydrolysis results in release of DNA J and PI. 637 00:43:37,770 --> 00:43:39,780 So now what do we have? 638 00:43:39,780 --> 00:43:42,960 ATP's hydrolyzed, and now we have ADP 639 00:43:42,960 --> 00:43:46,390 bound in the nucleotide binding domain. 640 00:43:46,390 --> 00:43:47,670 And what do we see? 641 00:43:47,670 --> 00:43:49,440 The latch has closed-- 642 00:43:49,440 --> 00:43:51,330 open, closed. 643 00:43:51,330 --> 00:43:53,400 So like what we saw in the structures 644 00:43:53,400 --> 00:43:56,250 with those model polypeptides mound, 645 00:43:56,250 --> 00:44:01,750 we have the substrate clamped in this latch. 646 00:44:01,750 --> 00:44:05,160 So here we have a form of DNA K that 647 00:44:05,160 --> 00:44:10,020 binds the polypeptide with high affinity and slow exchange. 648 00:44:10,020 --> 00:44:15,960 So this state is considered to be long lived, 649 00:44:15,960 --> 00:44:18,210 on the order of 10 to 15 seconds. 650 00:44:18,210 --> 00:44:20,640 So the question is, if this is binding the polypeptide 651 00:44:20,640 --> 00:44:24,570 with high affinity and slow exchange, how do we release it? 652 00:44:24,570 --> 00:44:28,140 And that's where this nucleotide exchange factor 653 00:44:28,140 --> 00:44:30,430 GrpE comes into play. 654 00:44:30,430 --> 00:44:32,610 So here comes along GrpE. 655 00:44:32,610 --> 00:44:34,250 GrpE binds. 656 00:44:34,250 --> 00:44:38,340 GrpE binding results in release of ADP 657 00:44:38,340 --> 00:44:40,620 from the nucleotide binding domain. 658 00:44:40,620 --> 00:44:44,562 So GrpE is inserting its beta sheet into that clef, 659 00:44:44,562 --> 00:44:46,020 and it looks like something else is 660 00:44:46,020 --> 00:44:50,580 happening with that long alpha helix to facilitate this. 661 00:44:50,580 --> 00:44:53,770 But this was drawn before that 2015 study, 662 00:44:53,770 --> 00:44:55,980 so we just see it interacting here. 663 00:44:55,980 --> 00:44:57,870 But imagine that this region here 664 00:44:57,870 --> 00:45:02,580 is maybe interacting down here and doing something 665 00:45:02,580 --> 00:45:05,280 to facilitate peptide release. 666 00:45:05,280 --> 00:45:07,020 So now what? 667 00:45:07,020 --> 00:45:09,840 No nucleotides bound according to this model. 668 00:45:09,840 --> 00:45:13,200 Since the ADP is released, ATP binding 669 00:45:13,200 --> 00:45:16,020 is facilitated so ATP can bind. 670 00:45:16,020 --> 00:45:16,830 And what do we see? 671 00:45:16,830 --> 00:45:19,830 There's release of the peptide, release of GrpE, 672 00:45:19,830 --> 00:45:23,340 and this cycle can start over again. 673 00:45:23,340 --> 00:45:27,030 So effectively, the release of ADP 674 00:45:27,030 --> 00:45:33,570 is accelerated about 5,000 fold from the action of GrpE. 675 00:45:33,570 --> 00:45:36,630 And so GrpE is called a thermo sensor 676 00:45:36,630 --> 00:45:39,390 and can begin to think about why that might be. 677 00:45:39,390 --> 00:45:42,540 If, say, there's condition of heat shock or stress, 678 00:45:42,540 --> 00:45:46,980 maybe the cell wants DNA K to be able to hold on 679 00:45:46,980 --> 00:45:49,530 to this polypeptide rather than release it. 680 00:45:49,530 --> 00:45:53,680 So GrpE won't be doing its job under those conditions. 681 00:45:59,060 --> 00:46:03,220 So another example of ATP binding and hydrolysis 682 00:46:03,220 --> 00:46:08,110 modulating activity of these chaperones. 683 00:46:08,110 --> 00:46:14,980 So we need to think about what are the substrates for DNA K J, 684 00:46:14,980 --> 00:46:18,340 and what is the chaperone system doing? 685 00:46:18,340 --> 00:46:22,300 So we define possibilities as foldases-- 686 00:46:22,300 --> 00:46:24,370 like what we saw with groEL-- 687 00:46:24,370 --> 00:46:29,690 holdases, unfoldases, what's happening here? 688 00:46:29,690 --> 00:46:36,940 And so in thinking about the in vitro substrates, what 689 00:46:36,940 --> 00:46:41,040 are the experiments we're going to do? 690 00:46:41,040 --> 00:46:43,150 Or sorry, in vivo substrates. 691 00:47:05,030 --> 00:47:08,930 So can we take the method used for groEL, groES, 692 00:47:08,930 --> 00:47:10,115 and adapt it to this system? 693 00:47:18,250 --> 00:47:20,350 Are you convinced that method was useful, 694 00:47:20,350 --> 00:47:22,000 or are you down on that method? 695 00:47:24,478 --> 00:47:26,020 AUDIENCE: It can probably be adapted. 696 00:47:26,020 --> 00:47:28,800 ELIZABETH NOLAN: Yeah, right, it can be adapted. 697 00:47:28,800 --> 00:47:37,880 So can imagine again going to do a pulse chase here, 698 00:47:37,880 --> 00:47:40,230 and can imagine the same experiments 699 00:47:40,230 --> 00:47:45,210 where we have our E coli with no methionine to deplete. 700 00:47:48,270 --> 00:47:52,140 We can pulse with radio labeled methionine-- 701 00:47:52,140 --> 00:47:55,140 again, 15 seconds, 30 degrees Celsius-- 702 00:47:58,120 --> 00:48:01,500 to let us see newly synthesized polypeptides. 703 00:48:01,500 --> 00:48:03,180 And this gives us a way to ask what 704 00:48:03,180 --> 00:48:09,240 newly synthesized polypeptides did DNA K and J act on. 705 00:48:09,240 --> 00:48:17,890 Then we can chase with excess unlabeled methionine 706 00:48:17,890 --> 00:48:18,700 for 10 minutes. 707 00:48:24,080 --> 00:48:32,685 And again, can take samples at varying times. 708 00:48:36,830 --> 00:48:37,805 Do rapid lysis. 709 00:48:42,190 --> 00:48:45,550 And in this case, rather than using EDTA to quench, 710 00:48:45,550 --> 00:48:49,240 what they did is do rapid ATP removal 711 00:48:49,240 --> 00:48:53,380 by adding an ATPase here. 712 00:48:53,380 --> 00:48:58,420 So just to realize that there's theme and variations in terms 713 00:48:58,420 --> 00:48:59,620 of how you can quench these. 714 00:49:12,590 --> 00:49:15,200 So what do they find? 715 00:49:15,200 --> 00:49:20,540 And we'll go over the data in more detail starting on Monday. 716 00:49:20,540 --> 00:49:23,600 And what do they need to do to find that? 717 00:49:23,600 --> 00:49:27,410 So in this case, they need an antibody to DNA K 718 00:49:27,410 --> 00:49:29,900 if there's going to be an immuno precipitation, right? 719 00:49:40,120 --> 00:49:44,800 So in these experiments, effectively we're 720 00:49:44,800 --> 00:49:46,190 to this point. 721 00:49:46,190 --> 00:49:50,140 They immunoprecipitated with their DNA K antibody. 722 00:49:50,140 --> 00:49:52,270 Of course, the specificity of this antibody 723 00:49:52,270 --> 00:49:55,570 needed to be studied, and then they 724 00:49:55,570 --> 00:50:00,190 used SDS-PAGE to analyze the immunoprecipitates. 725 00:50:00,190 --> 00:50:05,680 And so what we'll see when we discuss the data next time, 726 00:50:05,680 --> 00:50:07,630 the experiments were analogous to what 727 00:50:07,630 --> 00:50:10,600 was done with groEL, groES, but a few differences. 728 00:50:10,600 --> 00:50:13,600 They were less sophisticated in terms of the approach. 729 00:50:13,600 --> 00:50:19,780 So they use just standard 1D STS page rather than 2D, 730 00:50:19,780 --> 00:50:22,360 and it didn't go through the process 731 00:50:22,360 --> 00:50:25,180 of doing trypsin [? digestion ?] mass spec 732 00:50:25,180 --> 00:50:26,920 to identify the polypeptide. 733 00:50:26,920 --> 00:50:29,300 So it's more of a qualitative look. 734 00:50:29,300 --> 00:50:31,540 But we're going to ask starting on Monday 735 00:50:31,540 --> 00:50:34,060 what did they learn from analyzing 736 00:50:34,060 --> 00:50:37,798 these gels about the substrate scope of DNA K J? 737 00:50:37,798 --> 00:50:39,340 And then we have to ask the question, 738 00:50:39,340 --> 00:50:41,470 how does that help our understanding 739 00:50:41,470 --> 00:50:43,870 in terms of the type of chaperone activity 740 00:50:43,870 --> 00:50:46,360 that's occurring? 741 00:50:46,360 --> 00:50:48,190 So with that, I'll close. 742 00:50:48,190 --> 00:50:51,790 We'll end the chaperone unit with those experiments 743 00:50:51,790 --> 00:50:53,470 on Monday, and then we'll transition 744 00:50:53,470 --> 00:50:59,730 into module 3, the proteasome and degradation chambers.