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 00:00:26,317 --> 00:00:28,900 ELIZABETH NOLAN: So we're going to spend the first few minutes 9 00:00:28,900 --> 00:00:34,000 finishing up our discussions of the evolved orthogonal ribosome 10 00:00:34,000 --> 00:00:37,290 called Ribo-X and experiments that were done to characterize 11 00:00:37,290 --> 00:00:38,980 it and determine whether it did a better 12 00:00:38,980 --> 00:00:44,440 job at allowing the tRNA to suppress the amber stop code. 13 00:00:44,440 --> 00:00:47,470 And then we'll be transitioning into module 2. 14 00:00:47,470 --> 00:00:50,020 So we'll be starting protein folding today 15 00:00:50,020 --> 00:00:54,190 and be spending the next couple of days on that area 16 00:00:54,190 --> 00:00:56,770 and thinking about some of the macromolecular machines 17 00:00:56,770 --> 00:01:00,400 and other proteins involved. 18 00:01:00,400 --> 00:01:03,730 So just as a recap, where we left off last time 19 00:01:03,730 --> 00:01:06,220 with thinking about incorporating 20 00:01:06,220 --> 00:01:10,300 unnatural amino acids using a new ribosome. 21 00:01:10,300 --> 00:01:13,630 And so we discussed how an orthogonal ribosome 22 00:01:13,630 --> 00:01:17,350 was designed that binds to orthogonal mRNA. 23 00:01:17,350 --> 00:01:21,040 And then the idea was, can this orthogonal ribosome 24 00:01:21,040 --> 00:01:26,140 be improved, the immunogenicity and selection, 25 00:01:26,140 --> 00:01:30,640 to get a new orthogonal ribosome, where suppression 26 00:01:30,640 --> 00:01:34,060 of the amber stop codon by the tRNA 27 00:01:34,060 --> 00:01:37,990 is favored over termination by release factor 1? 28 00:01:37,990 --> 00:01:41,780 So we talked about this issue with really 29 00:01:41,780 --> 00:01:46,300 factor 1 causing truncated protein phenotypes. 30 00:01:46,300 --> 00:01:50,170 And so where we left off was in a series 31 00:01:50,170 --> 00:01:52,270 of three types of experiments to look 32 00:01:52,270 --> 00:01:54,880 at how well this Ribo-X works. 33 00:01:54,880 --> 00:01:57,370 So we looked at protein yield to ask, 34 00:01:57,370 --> 00:01:59,620 is it translating polypeptide, as well 35 00:01:59,620 --> 00:02:02,020 as the starting orthogonal ribosome. 36 00:02:02,020 --> 00:02:04,940 And then the next experiment we were looking at, 37 00:02:04,940 --> 00:02:07,360 and where we left off with, was experiment 38 00:02:07,360 --> 00:02:13,720 2, which was the question of amino acid incorporation. 39 00:02:27,520 --> 00:02:32,130 And so what was done, just in recap, 40 00:02:32,130 --> 00:02:37,900 is that a GST maltose-binding protein, MBP, fusion 41 00:02:37,900 --> 00:02:40,270 protein was designed. 42 00:02:40,270 --> 00:02:50,830 And recall that this protein had a protease cleavage site 43 00:02:50,830 --> 00:02:59,780 and that the GST portion contained cysteine and MBP does 44 00:02:59,780 --> 00:03:01,190 not contain cysteine. 45 00:03:04,880 --> 00:03:09,440 So the idea was to use radio labeled 46 00:03:09,440 --> 00:03:17,390 cysteine as a probe for misincorporation of cysteine 47 00:03:17,390 --> 00:03:20,750 into maltose-binding protein. 48 00:03:20,750 --> 00:03:24,650 So in terms of the actual experiment, what was done, 49 00:03:24,650 --> 00:03:29,330 imagine that we express this fusion. 50 00:03:33,730 --> 00:03:37,300 In the presence of the radio labelled cysteine, 51 00:03:37,300 --> 00:03:38,365 we use a protease. 52 00:03:42,570 --> 00:03:44,010 And in this case, it was thrombin. 53 00:03:47,410 --> 00:03:55,390 This protease gives us two fragments, GST plus MBP. 54 00:03:55,390 --> 00:03:58,420 And these two proteins have different size, 55 00:03:58,420 --> 00:03:59,530 so we can separate. 56 00:04:06,410 --> 00:04:08,045 So just doing SDS page. 57 00:04:12,470 --> 00:04:16,670 And ask, where did the proteins migrate on the gel, 58 00:04:16,670 --> 00:04:18,769 and then where do we see radioactivity? 59 00:04:21,560 --> 00:04:24,440 And so the data are shown here that were 60 00:04:24,440 --> 00:04:26,270 reported by the authors. 61 00:04:26,270 --> 00:04:28,710 So what are we looking at? 62 00:04:28,710 --> 00:04:31,220 We're looking at, on the left, the Coomassie 63 00:04:31,220 --> 00:04:33,920 stain for total protein. 64 00:04:33,920 --> 00:04:40,640 And then on the right, we're looking at radioactivity here. 65 00:04:40,640 --> 00:04:46,850 So what do we have here, in terms of the lanes? 66 00:04:46,850 --> 00:04:48,560 They've done some labeling to help us. 67 00:04:48,560 --> 00:04:53,000 So maltose-binding protein is running up here. 68 00:04:53,000 --> 00:04:56,620 GST is running down here. 69 00:04:56,620 --> 00:04:59,720 And we have four different conditions. 70 00:04:59,720 --> 00:05:00,710 What do we see? 71 00:05:00,710 --> 00:05:05,990 We have, in lane 1, Ribo-X is what's used. 72 00:05:05,990 --> 00:05:08,150 In lane 2, we have a system where 73 00:05:08,150 --> 00:05:11,480 it's the initial orthogonal ribosome that was not involved. 74 00:05:11,480 --> 00:05:14,480 So that's our point of comparison. 75 00:05:14,480 --> 00:05:18,080 In lane 3, we have the native ribosome only. 76 00:05:18,080 --> 00:05:23,030 And in the 4, lane 4, a control for no gene. 77 00:05:23,030 --> 00:05:25,000 So what do we see? 78 00:05:31,357 --> 00:05:33,313 AUDIENCE: All the ribosomes look like they're 79 00:05:33,313 --> 00:05:37,800 doing the same thing, except for when there's no gene. 80 00:05:37,800 --> 00:05:39,850 ELIZABETH NOLAN: Right, no gene to translate. 81 00:05:39,850 --> 00:05:40,350 Yeah. 82 00:05:40,350 --> 00:05:42,080 So why do we come to that conclusion, 83 00:05:42,080 --> 00:05:44,140 they're all doing the same thing? 84 00:05:44,140 --> 00:05:47,590 What we see on the left here, in the Coomassie stain, 85 00:05:47,590 --> 00:05:51,820 is that in lanes 1 through 3, we see a band for MVP, 86 00:05:51,820 --> 00:05:55,390 and they're all similar, in terms of intensity. 87 00:05:55,390 --> 00:06:01,130 And likewise, for GST, we see a band, all similar intensity. 88 00:06:01,130 --> 00:06:05,380 So it looks like, in all cases, a similar amount of protein 89 00:06:05,380 --> 00:06:10,420 is being synthesized, and that this protease cleavage worked 90 00:06:10,420 --> 00:06:11,413 in a comparable manner. 91 00:06:11,413 --> 00:06:13,580 AUDIENCE: Do you ever have different RNAs, depending 92 00:06:13,580 --> 00:06:15,288 on whether or not the orthogonal ribosome 93 00:06:15,288 --> 00:06:17,320 versus the native ribosome, and do you 94 00:06:17,320 --> 00:06:19,850 worry about how that might influence this experiment? 95 00:06:19,850 --> 00:06:21,058 ELIZABETH NOLAN: Yeah, right. 96 00:06:21,058 --> 00:06:23,930 The native ribosome is not going to bind to the orthogonal mRNA, 97 00:06:23,930 --> 00:06:25,340 so you need to give a plasma that 98 00:06:25,340 --> 00:06:28,220 has a ribosome binding site compatible 99 00:06:28,220 --> 00:06:29,780 with the native ribosome. 100 00:06:29,780 --> 00:06:33,440 And yes, it's possible that native ribosome 101 00:06:33,440 --> 00:06:35,270 won't work as well. 102 00:06:35,270 --> 00:06:36,560 Or reverse. 103 00:06:36,560 --> 00:06:39,920 I'd say reverse is more likely, that one of these mutants 104 00:06:39,920 --> 00:06:41,840 won't work as well. 105 00:06:41,840 --> 00:06:44,310 But that doesn't appear to be the case here. 106 00:06:44,310 --> 00:06:48,980 So what does the gel on the right show? 107 00:06:48,980 --> 00:06:54,280 So where do we see radioactivity? 108 00:06:54,280 --> 00:06:55,780 AUDIENCE: Mostly at GST, but then it 109 00:06:55,780 --> 00:06:57,775 looks like there's a lot of background. 110 00:06:57,775 --> 00:06:58,692 ELIZABETH NOLAN: Yeah. 111 00:06:58,692 --> 00:07:01,550 Do we expect background? 112 00:07:01,550 --> 00:07:03,910 Where might the background come from? 113 00:07:03,910 --> 00:07:06,970 Right, so we see a strong band here, 114 00:07:06,970 --> 00:07:10,180 and this is at the same places where we see GST. 115 00:07:10,180 --> 00:07:11,710 So that's a good indication. 116 00:07:11,710 --> 00:07:13,750 We know that there's cysteine and GST, 117 00:07:13,750 --> 00:07:18,730 so we should see a band here. 118 00:07:18,730 --> 00:07:21,640 So what about background and what about MBP? 119 00:07:27,800 --> 00:07:30,300 AUDIENCE: You're going to have some misappropriation in MBP. 120 00:07:30,300 --> 00:07:33,410 It's very much falsely compared with GST. 121 00:07:33,410 --> 00:07:36,150 But a little band in MBP could be some amino acid 122 00:07:36,150 --> 00:07:37,560 misappropriation. 123 00:07:37,560 --> 00:07:40,410 ELIZABETH NOLAN: OK, is that band MBP? 124 00:07:40,410 --> 00:07:42,310 Do we know that definitively in this gel? 125 00:07:42,310 --> 00:07:44,560 AUDIENCE: Not necessarily because the Coomassie band's 126 00:07:44,560 --> 00:07:46,185 a little wider, so it can be a slightly 127 00:07:46,185 --> 00:07:47,570 smaller molecular weight then. 128 00:07:47,570 --> 00:07:48,790 ELIZABETH NOLAN: Right. 129 00:07:48,790 --> 00:07:52,740 There's many species in these gels. 130 00:07:52,740 --> 00:07:56,550 It isn't just two bands, one for GST and one for MBP. 131 00:07:56,550 --> 00:08:00,870 So where did these other bands come from? 132 00:08:00,870 --> 00:08:03,390 Could it be from the initial protein purification 133 00:08:03,390 --> 00:08:05,190 and there's some contaminants? 134 00:08:05,190 --> 00:08:07,380 Could it be a result of the thrombin cleavage, 135 00:08:07,380 --> 00:08:10,230 and maybe thrombin cut at some places other 136 00:08:10,230 --> 00:08:11,670 than just this cleavage site? 137 00:08:11,670 --> 00:08:14,880 So that's preferred, but maybe it can cut some other places. 138 00:08:14,880 --> 00:08:18,400 So there's other species in the gel. 139 00:08:18,400 --> 00:08:21,550 And cysteine comes up in other proteins, 140 00:08:21,550 --> 00:08:23,940 so it makes sense that there'd be some background 141 00:08:23,940 --> 00:08:28,770 to say this protein wasn't 99%, 100% pure, 142 00:08:28,770 --> 00:08:30,810 or maybe from cleavage there. 143 00:08:30,810 --> 00:08:35,010 In terms of this little band, is that MBP or not? 144 00:08:35,010 --> 00:08:35,940 A little hard to say. 145 00:08:35,940 --> 00:08:39,299 Where they placed the arrow indicates maybe not, right? 146 00:08:39,299 --> 00:08:43,205 But what could you do to find that out? 147 00:08:43,205 --> 00:08:45,093 AUDIENCE: Western. 148 00:08:45,093 --> 00:08:46,010 ELIZABETH NOLAN: Yeah. 149 00:08:46,010 --> 00:08:47,260 So maybe a Western Blot. 150 00:08:47,260 --> 00:08:49,330 If there's an antibody, you could run 151 00:08:49,330 --> 00:08:51,370 something less concentrated. 152 00:08:51,370 --> 00:08:53,290 You could do mass spec. 153 00:08:53,290 --> 00:08:56,140 There's options if you wanted to track that down. 154 00:08:56,140 --> 00:09:00,040 But the bottom line is if we compare the radioactivity here 155 00:09:00,040 --> 00:09:02,290 to what's seen here, there's much more 156 00:09:02,290 --> 00:09:05,680 with GST, which is what we would expect. 157 00:09:05,680 --> 00:09:07,720 And so through some further analysis, 158 00:09:07,720 --> 00:09:10,780 they conclude the error frequency is less than 1 159 00:09:10,780 --> 00:09:15,270 in 10 to the 3 for Ribo-X, at least for cysteine. 160 00:09:15,270 --> 00:09:18,280 So what's the limitation of this experiment, from the standpoint 161 00:09:18,280 --> 00:09:22,040 of misincorporation, which is what I asked you to think about 162 00:09:22,040 --> 00:09:23,310 in closing last time? 163 00:09:26,100 --> 00:09:27,600 AUDIENCE: What you're testing for is 164 00:09:27,600 --> 00:09:29,150 cysteine in this incorporation. 165 00:09:29,150 --> 00:09:30,900 ELIZABETH NOLAN: Only testing for cysteine 166 00:09:30,900 --> 00:09:32,790 in this incorporation. 167 00:09:32,790 --> 00:09:35,750 So we don't really know about other amino acids. 168 00:09:35,750 --> 00:09:38,430 So this is good news, but there's 169 00:09:38,430 --> 00:09:41,370 other information we're not getting from this experiment 170 00:09:41,370 --> 00:09:43,140 there. 171 00:09:43,140 --> 00:09:45,130 So is it possible that other amino acids 172 00:09:45,130 --> 00:09:46,110 are misincorporated? 173 00:09:46,110 --> 00:09:50,020 It would take some additional experiments to get at that. 174 00:09:50,020 --> 00:09:51,600 So the last experiment we're going 175 00:09:51,600 --> 00:09:55,410 to look at is asking the question, 176 00:09:55,410 --> 00:09:58,140 does Ribo-X actually do a better job 177 00:09:58,140 --> 00:10:01,530 than the progenitor or o-ribosome? 178 00:10:01,530 --> 00:10:07,710 So what we want to look is at the efficiency of suppression 179 00:10:07,710 --> 00:10:08,780 of the amber stop codon. 180 00:10:22,370 --> 00:10:24,680 So last time, we talked about how 181 00:10:24,680 --> 00:10:27,230 one limitation of the Schultz method 182 00:10:27,230 --> 00:10:31,640 is that there's a truncated phenotype because RF one enters 183 00:10:31,640 --> 00:10:34,130 the A state, rather than the tRNA. 184 00:10:34,130 --> 00:10:36,710 So in order to look at this, they 185 00:10:36,710 --> 00:10:40,490 continued to use this type of fusion. 186 00:10:40,490 --> 00:10:45,380 But instead of having a protease cleavage site here, 187 00:10:45,380 --> 00:10:52,010 they stuck to the amber stop codon in between GST and MBP. 188 00:10:52,010 --> 00:10:55,220 So they made two different plasmids, 189 00:10:55,220 --> 00:11:03,710 so GST, a plasma encoding, GST, the stop, and malE. 190 00:11:03,710 --> 00:11:07,190 So this is the gene named for MBP. 191 00:11:07,190 --> 00:11:14,810 So if this gets translated, the question is, 192 00:11:14,810 --> 00:11:23,450 do we get the GST-MBP fusion or GST? 193 00:11:23,450 --> 00:11:27,080 Because if the tRNA does its job, 194 00:11:27,080 --> 00:11:29,000 polypeptide synthesis will continue 195 00:11:29,000 --> 00:11:31,060 and we'll get the fusion protein. 196 00:11:31,060 --> 00:11:34,310 If release factor 1 ends up here at this position, 197 00:11:34,310 --> 00:11:38,600 we get termination and only GST. 198 00:11:38,600 --> 00:11:42,470 And they also made an additional construct. 199 00:11:42,470 --> 00:11:45,110 So in addition to asking, what happens 200 00:11:45,110 --> 00:11:49,100 if there is a codon for one unnatural amino acid, what 201 00:11:49,100 --> 00:11:50,690 happens if there's two? 202 00:11:50,690 --> 00:11:52,970 So recall, with the Schultz method, 203 00:11:52,970 --> 00:11:56,630 what we saw last time is that when attempts were made 204 00:11:56,630 --> 00:11:59,920 to incorporate two unnatural amino acids in one polypeptide, 205 00:11:59,920 --> 00:12:03,770 the efficiency went down to below 1% there. 206 00:12:03,770 --> 00:12:11,940 So what if two of these are here? 207 00:12:11,940 --> 00:12:19,410 Again, the question is, when these get translated, 208 00:12:19,410 --> 00:12:24,570 do we got GST-MBP or GST? 209 00:12:30,340 --> 00:12:34,480 And as we can see from the gel, we 210 00:12:34,480 --> 00:12:37,510 can differentiate pieces by size. 211 00:12:37,510 --> 00:12:38,650 OK. 212 00:12:38,650 --> 00:12:43,450 So in addition to comparing these, what was done 213 00:12:43,450 --> 00:12:46,630 is in both sets of experiments, there 214 00:12:46,630 --> 00:12:57,210 was a comparison of the Schultz method 215 00:12:57,210 --> 00:13:02,070 we discussed first, and Ribo-X here. 216 00:13:05,380 --> 00:13:14,410 And just in terms of size, so GST-MBP is about 70 kilodaltons 217 00:13:14,410 --> 00:13:20,180 and GST is about 20 kilodaltons, 26. 218 00:13:20,180 --> 00:13:23,260 So easy to separate on the gel. 219 00:13:26,230 --> 00:13:27,385 So what are the data? 220 00:13:32,340 --> 00:13:37,010 And what was the unnatural amino acid employed? 221 00:13:37,010 --> 00:13:42,830 So they ended up using an unnatural amino acid called 222 00:13:42,830 --> 00:13:48,680 BPA, which is a benzophenone, so a crosslinker. 223 00:14:02,790 --> 00:14:03,290 Here. 224 00:14:06,680 --> 00:14:10,610 And so I'm going to write up some details that came out 225 00:14:10,610 --> 00:14:16,280 from the gel on the board because there's a lot of things 226 00:14:16,280 --> 00:14:17,930 to navigate in that gel. 227 00:14:27,300 --> 00:14:30,720 So effectively, what are the comparisons we want to make? 228 00:14:30,720 --> 00:14:34,695 So in lane 3, the method we have is the Schultz method. 229 00:14:37,900 --> 00:14:44,250 There is one codon for incorporating 230 00:14:44,250 --> 00:14:46,170 one unnatural amino acid. 231 00:14:46,170 --> 00:14:50,310 And what they see is they get about 24% efficiency 232 00:14:50,310 --> 00:14:53,700 of full length fusion protein. 233 00:14:53,700 --> 00:14:58,950 In lane 5, we have the Shultz method with two here, 234 00:14:58,950 --> 00:15:03,750 and we get about 1% efficiency 235 00:15:03,750 --> 00:15:09,510 In lane 7, analyzing the orthogonal 236 00:15:09,510 --> 00:15:13,800 ribosome Ribo-X, and one. 237 00:15:13,800 --> 00:15:20,490 And what's seen is 64% efficiency, so quite 238 00:15:20,490 --> 00:15:21,870 an improvement there. 239 00:15:21,870 --> 00:15:24,840 And then in lane 9, what we have is 240 00:15:24,840 --> 00:15:30,650 Ribo-X with two and an efficiency of 22% here. 241 00:15:30,650 --> 00:15:39,690 OK so here, we're looking at the wild type ribosome, 242 00:15:39,690 --> 00:15:45,360 and here, we're looking at the orthogonal ribosome, 243 00:15:45,360 --> 00:15:52,630 orthogonal mRNA with the two mutations we saw before. 244 00:15:52,630 --> 00:15:58,200 So these values come up from quantification of the data 245 00:15:58,200 --> 00:15:59,580 here. 246 00:15:59,580 --> 00:16:06,030 So you can convince yourself by comparing the bands for GST, 247 00:16:06,030 --> 00:16:08,880 resulting from truncated phenotype translation 248 00:16:08,880 --> 00:16:14,910 termination and the bands for the fusion protein GST-MBP. 249 00:16:14,910 --> 00:16:17,520 So showing that there was successful suppression 250 00:16:17,520 --> 00:16:20,370 of the amber stop codon here. 251 00:16:20,370 --> 00:16:24,150 So what are the major conclusions? 252 00:16:24,150 --> 00:16:27,340 The major conclusion is that, at least with this system, 253 00:16:27,340 --> 00:16:31,650 what we see is that Ribo-X has minimized 254 00:16:31,650 --> 00:16:35,610 this truncated peptide phenotype compared to the wild type 255 00:16:35,610 --> 00:16:38,970 ribosome, and that it's been possible to, 256 00:16:38,970 --> 00:16:42,450 basically, diverge the decoding properties 257 00:16:42,450 --> 00:16:45,780 of the orthogonal ribosome from the indigenous cellular 258 00:16:45,780 --> 00:16:46,976 machinery. 259 00:16:46,976 --> 00:16:49,025 AUDIENCE: I'm trying to understand you right. 260 00:16:49,025 --> 00:16:51,960 So the percent here, that's the percent 261 00:16:51,960 --> 00:16:53,510 of the total expressed protein? 262 00:16:53,510 --> 00:16:58,890 Oh, so the other 99% would have been the GST only? 263 00:16:58,890 --> 00:16:59,980 ELIZABETH NOLAN: Yeah. 264 00:16:59,980 --> 00:17:02,610 So here, for instance, if we take a look at lane-- 265 00:17:02,610 --> 00:17:05,480 let's compare lanes 3 and lane 5. 266 00:17:05,480 --> 00:17:08,460 So here's lane 3, and what do we see? 267 00:17:08,460 --> 00:17:12,270 So in here, we have incorporation 268 00:17:12,270 --> 00:17:13,560 of one natural amino acid. 269 00:17:13,560 --> 00:17:17,790 And we see that there is a band for the fusion protein 270 00:17:17,790 --> 00:17:18,687 and there's a band-- 271 00:17:18,687 --> 00:17:20,520 let me make sure I'm in the right lane, lane 272 00:17:20,520 --> 00:17:23,540 3-- and a band for GST itself. 273 00:17:23,540 --> 00:17:25,260 And the intensity of this band is 274 00:17:25,260 --> 00:17:27,000 greater than the intensity of that band, 275 00:17:27,000 --> 00:17:29,070 and you can imagine doing quantitation, 276 00:17:29,070 --> 00:17:31,380 whereas if we look at lane 5, where we're 277 00:17:31,380 --> 00:17:33,930 trying to incorporate two by this method, 278 00:17:33,930 --> 00:17:37,350 we see a band for GST. 279 00:17:37,350 --> 00:17:38,940 And what do we see up here? 280 00:17:38,940 --> 00:17:41,550 Very little. 281 00:17:41,550 --> 00:17:45,120 If we look at lane 7, we're seeing 64%. 282 00:17:45,120 --> 00:17:50,310 Lane 7 here, we see that we have this band for the GST-MBP 283 00:17:50,310 --> 00:17:55,590 fusion and a weaker band for GST alone there. 284 00:17:55,590 --> 00:18:00,630 So percent efficiency, percent of the total. 285 00:18:00,630 --> 00:18:03,690 So there's other things happening in this field. 286 00:18:03,690 --> 00:18:06,300 So the Schultz method and these orthogonal ribosomes 287 00:18:06,300 --> 00:18:07,530 are two examples. 288 00:18:07,530 --> 00:18:11,250 One thing that came up after this work with Ribo-X 289 00:18:11,250 --> 00:18:15,240 was to design ribosomes that can use quadrupling codons, 290 00:18:15,240 --> 00:18:17,980 rather than triplets. 291 00:18:17,980 --> 00:18:20,970 So a lot of creativity and things 292 00:18:20,970 --> 00:18:23,290 to look up if you're curious. 293 00:18:23,290 --> 00:18:26,740 But with that, we're going to close the translation module. 294 00:18:26,740 --> 00:18:28,110 We will not leave the ribosome. 295 00:18:28,110 --> 00:18:33,000 It will keep popping up throughout modules 2 and 3. 296 00:18:33,000 --> 00:18:37,380 But we're going to move into what happens to a polypeptide 297 00:18:37,380 --> 00:18:39,690 as it leaves the ribosome. 298 00:18:39,690 --> 00:18:42,970 So how does it get its native fold? 299 00:18:42,970 --> 00:18:46,440 And so what happens to nascent polypeptides 300 00:18:46,440 --> 00:18:50,130 emerging from the ribosome, and how do polypeptides fold? 301 00:18:50,130 --> 00:18:54,760 And so there's reading posted for module 2 on Stellar 302 00:18:54,760 --> 00:18:57,150 and listed here, one required paper, 303 00:18:57,150 --> 00:19:00,180 which is a really wonderful review that 304 00:19:00,180 --> 00:19:03,040 came out about two years ago. 305 00:19:03,040 --> 00:19:05,010 So let's think about folding. 306 00:19:07,860 --> 00:19:14,970 And as a point to thinking about that, 307 00:19:14,970 --> 00:19:18,390 let's think about our ribosome. 308 00:19:18,390 --> 00:19:23,540 And there's some emerging polypeptide chain, 309 00:19:23,540 --> 00:19:25,185 so the nascent polypeptide. 310 00:19:33,090 --> 00:19:36,510 So what happens to this polypeptide? 311 00:19:36,510 --> 00:19:39,150 And the first thing to keep in mind is something 312 00:19:39,150 --> 00:19:42,390 we need to think about, is where is this polypeptide destined 313 00:19:42,390 --> 00:19:43,410 to go? 314 00:19:43,410 --> 00:19:46,980 Is this a polypeptide that will be in the cytoplasm? 315 00:19:46,980 --> 00:19:49,770 Is this a polypeptide that will become 316 00:19:49,770 --> 00:19:54,010 a membrane protein, or part of the secretory system? 317 00:19:54,010 --> 00:20:06,260 And so we can think about cytoplasmic protein 318 00:20:06,260 --> 00:20:22,490 versus membrane proteins, or a new karyote secretory here. 319 00:20:22,490 --> 00:20:27,175 And so we're going to focus this module 320 00:20:27,175 --> 00:20:28,550 in terms of thinking about what's 321 00:20:28,550 --> 00:20:30,380 happening in the cytoplasm. 322 00:20:30,380 --> 00:20:32,470 We might touch upon this if there's time, 323 00:20:32,470 --> 00:20:34,610 but I think there won't be. 324 00:20:34,610 --> 00:20:48,380 So the cytoplasmic proteins are folded by chaperones 325 00:20:48,380 --> 00:20:51,350 that they can come into contact with as they're 326 00:20:51,350 --> 00:20:53,870 emerging from the ribosome, or also 327 00:20:53,870 --> 00:20:56,150 after the polypeptide is released. 328 00:20:56,150 --> 00:20:58,670 AUDIENCE: Do extracellular matrix proteins 329 00:20:58,670 --> 00:21:01,450 fall into either of these categories? 330 00:21:01,450 --> 00:21:03,080 ELIZABETH NOLAN: I actually don't know. 331 00:21:03,080 --> 00:21:07,297 Joanne, where do extracellular matrix proteins fall? 332 00:21:07,297 --> 00:21:09,130 JOANNE: Well, they get made inside the cell. 333 00:21:09,130 --> 00:21:10,060 ELIZABETH NOLAN: They're made inside the cell 334 00:21:10,060 --> 00:21:11,140 and then they have get shuttled. 335 00:21:11,140 --> 00:21:13,098 JOANNE: So you should go talk to Matt Shoulders 336 00:21:13,098 --> 00:21:17,466 because if you look at collagen, that's exactly [laughter] 337 00:21:17,466 --> 00:21:34,310 ELIZABETH NOLAN: OK, so these interact with a player 338 00:21:34,310 --> 00:21:42,290 called signal recognition particle, which 339 00:21:42,290 --> 00:21:56,660 allows for targeting to the membrane or endoplasmic 340 00:21:56,660 --> 00:22:01,940 reticulum in eukaryotes, and then folding can happen here. 341 00:22:01,940 --> 00:22:05,170 So we're going to be focused in the cytoplasm, just 342 00:22:05,170 --> 00:22:07,690 realize there's other machineries involved 343 00:22:07,690 --> 00:22:08,635 for membrane proteins. 344 00:22:11,580 --> 00:22:14,660 So here's just another view of our ribosome. 345 00:22:14,660 --> 00:22:17,540 We saw this early on in the ribosome unit. 346 00:22:17,540 --> 00:22:21,650 And we want to think about this exit tunnel and the emerging 347 00:22:21,650 --> 00:22:24,380 polypeptide chain. 348 00:22:24,380 --> 00:22:26,720 So it's the 50S subunit. 349 00:22:26,720 --> 00:22:31,820 And as we discussed before, this exit tunnel is long 350 00:22:31,820 --> 00:22:34,310 and it's also quite narrow and it's 351 00:22:34,310 --> 00:22:37,880 lined by both ribosomes RNA and proteins. 352 00:22:37,880 --> 00:22:40,550 And I know a few of you asked about the hydrophobic residues 353 00:22:40,550 --> 00:22:44,420 of proteins that line this tunnel after lecture 2. 354 00:22:44,420 --> 00:22:47,510 And the thing to keep in mind is that it's not all hydrophobic. 355 00:22:47,510 --> 00:22:48,950 There's also RNA there. 356 00:22:48,950 --> 00:22:51,322 There will also be other residues. 357 00:22:51,322 --> 00:22:53,780 And something just to think about, like can water molecules 358 00:22:53,780 --> 00:22:56,640 get in there, as well? 359 00:22:56,640 --> 00:22:59,420 So we see for instance, there's two proteins, 360 00:22:59,420 --> 00:23:02,990 L4 and L22, that line part of the tunnel. 361 00:23:02,990 --> 00:23:05,980 We have protein L23 at the exit. 362 00:23:05,980 --> 00:23:09,680 But there's also a lot of RNA there, so don't forget that. 363 00:23:09,680 --> 00:23:13,320 So a question, just to address early on, 364 00:23:13,320 --> 00:23:16,490 does protein folding occur in the exit tunnel? 365 00:23:16,490 --> 00:23:19,490 And I'd say this has been a bit of a controversial question 366 00:23:19,490 --> 00:23:25,310 over the years and there've been camps arguing 367 00:23:25,310 --> 00:23:27,576 both possibilities, yes or no. 368 00:23:27,576 --> 00:23:29,660 I think the thing to keep in mind 369 00:23:29,660 --> 00:23:32,900 is that the dimensions are limited. 370 00:23:32,900 --> 00:23:35,780 And although we can imagine some confirmation of flexibility 371 00:23:35,780 --> 00:23:38,180 and dynamics in this exit tunnel, 372 00:23:38,180 --> 00:23:40,520 it can't undergo some tremendous change 373 00:23:40,520 --> 00:23:43,850 to, say, accommodate something like ubiquitin 374 00:23:43,850 --> 00:23:46,200 that you saw early on. 375 00:23:46,200 --> 00:23:49,190 That doesn't just make sense. 376 00:23:49,190 --> 00:23:52,700 So is it possible for some alpha-helical fold to occur 377 00:23:52,700 --> 00:23:54,530 in this exit tunnel? 378 00:23:54,530 --> 00:23:55,820 Presumably. 379 00:23:55,820 --> 00:23:57,530 There is some work that indicates 380 00:23:57,530 --> 00:23:59,840 there's folding zones in the exit tunnel, 381 00:23:59,840 --> 00:24:02,870 so maybe some folding happens there. 382 00:24:02,870 --> 00:24:04,550 But really, the main conclusion is 383 00:24:04,550 --> 00:24:09,560 that most folding occurs outside of the ribosome 384 00:24:09,560 --> 00:24:15,470 and after the polypeptide emerges from the 50S here. 385 00:24:15,470 --> 00:24:18,830 So if we're thinking about most folding of polypeptides 386 00:24:18,830 --> 00:24:23,210 as occurring in the cytoplasm for cytoplasmic proteins, what 387 00:24:23,210 --> 00:24:26,940 we need to think about is that environment. 388 00:24:26,940 --> 00:24:30,050 And we learned in the introductory lectures, 389 00:24:30,050 --> 00:24:33,260 or had a reminder, that the cellular environment is 390 00:24:33,260 --> 00:24:34,520 very crowded. 391 00:24:34,520 --> 00:24:37,910 So we have this issue of macromolecular crowding. 392 00:24:37,910 --> 00:24:42,560 And in thinking about that, we need to ask the question, 393 00:24:42,560 --> 00:24:48,770 how does this emerging polypeptide fold 394 00:24:48,770 --> 00:24:53,030 to its native form in this type of environment? 395 00:24:53,030 --> 00:24:56,390 What type of machinery is there to help protect it? 396 00:24:56,390 --> 00:25:00,260 How is misfolding avoided and intermolecular 397 00:25:00,260 --> 00:25:03,305 molecular interactions that are non-productive avoided? 398 00:25:06,050 --> 00:25:09,200 So where are we going to go in this module? 399 00:25:09,200 --> 00:25:12,250 We're going to look at protein folding from both the 400 00:25:12,250 --> 00:25:17,120 in vitro test tube perspectives and also from in the cell. 401 00:25:17,120 --> 00:25:21,070 And so in thinking about protein folding in vitro, 402 00:25:21,070 --> 00:25:23,490 we'll discuss some of the seminal study, so 403 00:25:23,490 --> 00:25:25,630 Anfinsen's hypothesis and folding 404 00:25:25,630 --> 00:25:29,860 of ribonucelic A, Levinthal's paradox, which brings us 405 00:25:29,860 --> 00:25:31,990 to thinking about energy landscapes, 406 00:25:31,990 --> 00:25:34,780 and also touch upon some of the experimental methods that 407 00:25:34,780 --> 00:25:36,640 are employed. 408 00:25:36,640 --> 00:25:43,180 And then in terms of machines, we'll think about, largely, 409 00:25:43,180 --> 00:25:46,160 post-translational protein folding in the cytoplasm, 410 00:25:46,160 --> 00:25:51,280 so GroEL, GroES, DnaK and J. We'll also talk about a protein 411 00:25:51,280 --> 00:25:53,800 called trigger factor that associates 412 00:25:53,800 --> 00:25:58,140 with the ribosome, and those nascent polypeptide chains. 413 00:25:58,140 --> 00:25:58,930 OK. 414 00:25:58,930 --> 00:26:04,900 So these machineries fold soluble proteins, not 415 00:26:04,900 --> 00:26:06,340 membrane proteins. 416 00:26:06,340 --> 00:26:09,550 And I'd also like to point out, and again, we may or may not 417 00:26:09,550 --> 00:26:12,610 get to these systems, depending on time, 418 00:26:12,610 --> 00:26:16,420 but in addition to these chaperones and macromolecular 419 00:26:16,420 --> 00:26:17,890 machines involved in folding, there 420 00:26:17,890 --> 00:26:21,160 are classical enzymes that are really important. 421 00:26:21,160 --> 00:26:23,830 And these include enzymes that, say, 422 00:26:23,830 --> 00:26:28,480 isomerize prolene, also thyle oxidases and isomerases there. 423 00:26:31,910 --> 00:26:35,780 So what are our questions for this module? 424 00:26:35,780 --> 00:26:38,560 So why and how are proteins folded? 425 00:26:38,560 --> 00:26:41,570 And in terms of how, in the lab versus in 426 00:26:41,570 --> 00:26:47,210 the cell, classical enzymes and micromolecular machines. 427 00:26:47,210 --> 00:26:50,720 What happens when proteins are misfolded? 428 00:26:50,720 --> 00:26:53,750 How does protein folding relate to disease? 429 00:26:53,750 --> 00:26:57,300 What methods are employed to study these phenomenon? 430 00:26:57,300 --> 00:26:59,330 And for the case studies, we'll look at, 431 00:26:59,330 --> 00:27:03,230 in terms of cytoplasmic players, we 432 00:27:03,230 --> 00:27:06,710 want to understand, really, what are 433 00:27:06,710 --> 00:27:09,590 the structural properties of these different chaperones 434 00:27:09,590 --> 00:27:11,600 and their partners? 435 00:27:11,600 --> 00:27:15,270 How do their structures relate to function? 436 00:27:15,270 --> 00:27:18,600 How do they help peptides attain the native fold? 437 00:27:18,600 --> 00:27:21,680 And how good is our understanding of these systems? 438 00:27:21,680 --> 00:27:24,650 We'll see in the case of DnaK/J, it's actually pretty difficult 439 00:27:24,650 --> 00:27:28,550 to know what they're actually doing here. 440 00:27:28,550 --> 00:27:30,800 And really, what is the experimental basis 441 00:27:30,800 --> 00:27:31,950 for our understanding? 442 00:27:36,340 --> 00:27:41,590 If we just take an overview of folding and misfolding-- 443 00:27:41,590 --> 00:27:44,800 and this is diagram for a eukaryotic cell 444 00:27:44,800 --> 00:27:48,470 and from many, many different types of studies-- 445 00:27:48,470 --> 00:27:50,470 what do we see? 446 00:27:50,470 --> 00:27:53,860 So we see that some sort of biomolecule 447 00:27:53,860 --> 00:27:56,900 called chaperone keeps coming up again and again. 448 00:27:56,900 --> 00:28:00,250 So these are proteins that assist with folding, 449 00:28:00,250 --> 00:28:03,350 or unfolding, disaggregation. 450 00:28:03,350 --> 00:28:08,440 There's many possibilities for the trajectory of a protein 451 00:28:08,440 --> 00:28:09,610 here. 452 00:28:09,610 --> 00:28:12,400 So here, we see a nascent polypeptide 453 00:28:12,400 --> 00:28:14,590 emerging from the ribosome. 454 00:28:14,590 --> 00:28:20,540 And imagine that some folding intermediate is released. 455 00:28:20,540 --> 00:28:22,810 So this is not fully at the native fold, 456 00:28:22,810 --> 00:28:24,820 but it's somewhere along that pathway. 457 00:28:24,820 --> 00:28:26,230 What might happen? 458 00:28:26,230 --> 00:28:28,570 Right here, what we see is some chaperones 459 00:28:28,570 --> 00:28:32,710 allow this intermediate to form a native protein. 460 00:28:32,710 --> 00:28:34,630 But look, there can also be unfolding, 461 00:28:34,630 --> 00:28:37,300 and this could work its way back. 462 00:28:37,300 --> 00:28:42,550 This native protein could unfold to a misfolded state. 463 00:28:42,550 --> 00:28:45,160 We can think about remodeling, and maybe there's 464 00:28:45,160 --> 00:28:48,400 chaperones involved and taking this misfolded state back 465 00:28:48,400 --> 00:28:52,360 to an intermediate that's on a productive pathway. 466 00:28:52,360 --> 00:28:54,080 What happens here? 467 00:28:54,080 --> 00:28:56,350 Maybe there's some trouble, and rather 468 00:28:56,350 --> 00:28:59,110 than reaching its native fold, this intermediate 469 00:28:59,110 --> 00:29:01,010 ends up aggregating. 470 00:29:01,010 --> 00:29:03,580 It forms some sort of protein aggregate, 471 00:29:03,580 --> 00:29:06,010 and maybe that can form oligomers, 472 00:29:06,010 --> 00:29:08,260 or some sort of amyloid fibril, like what we hear 473 00:29:08,260 --> 00:29:10,780 about with Alzheimer's disease. 474 00:29:10,780 --> 00:29:12,610 Here, we see there's chaperones that 475 00:29:12,610 --> 00:29:15,970 can be involved in having disaggregate activity, 476 00:29:15,970 --> 00:29:18,430 and they can help in breaking down these aggregates 477 00:29:18,430 --> 00:29:21,920 and getting back to some productive place here. 478 00:29:21,920 --> 00:29:22,570 OK. 479 00:29:22,570 --> 00:29:27,370 So there's inherent complexity here 480 00:29:27,370 --> 00:29:31,720 and many players and relationships 481 00:29:31,720 --> 00:29:36,460 between protein misfolding and disease, just to be aware of. 482 00:29:36,460 --> 00:29:38,290 So we typically think about the protein 483 00:29:38,290 --> 00:29:42,730 fold providing function and protein misfolding 484 00:29:42,730 --> 00:29:44,990 can result in improper function. 485 00:29:44,990 --> 00:29:48,280 And there's many different types of improper function. 486 00:29:48,280 --> 00:29:50,120 It could be loss of function. 487 00:29:50,120 --> 00:29:52,070 It could be gain of function. 488 00:29:52,070 --> 00:29:54,190 It could be formation of some sort of aggregate 489 00:29:54,190 --> 00:29:58,370 that's deleterious to the cell for one reason or another. 490 00:29:58,370 --> 00:30:01,480 And if we just take a look, in terms of human diseases 491 00:30:01,480 --> 00:30:05,890 that are associated with protein misfolding, what do we see? 492 00:30:05,890 --> 00:30:10,510 So there's examples out there, like Alzheimer's, Parkinson's, 493 00:30:10,510 --> 00:30:14,080 familial ALS, and mad cow. 494 00:30:14,080 --> 00:30:17,980 So Alzheimer's disease is associated with formation 495 00:30:17,980 --> 00:30:20,720 of Abeta plaques in the brain. 496 00:30:20,720 --> 00:30:24,580 In Parkinson's there's a peptide called alpha-synnuclein 497 00:30:24,580 --> 00:30:28,390 that aggregates in familial ALS, also 498 00:30:28,390 --> 00:30:30,340 called Lou Gehrig's disease. 499 00:30:30,340 --> 00:30:34,690 There are single point mutations in an enzyme called 500 00:30:34,690 --> 00:30:36,970 superoxide dismutase that results 501 00:30:36,970 --> 00:30:41,080 in misfolding and some negative consequences there. 502 00:30:41,080 --> 00:30:43,280 And then misfolding of the prion protein. 503 00:30:43,280 --> 00:30:46,990 So a lot of these, in terms of neurological disorders. 504 00:30:46,990 --> 00:30:49,780 So in addition to fundamental studies, 505 00:30:49,780 --> 00:30:52,190 there's significant interest in understanding protein 506 00:30:52,190 --> 00:30:56,860 misfolding from the standpoint of disease and prevention. 507 00:30:56,860 --> 00:31:00,310 And I'll just note, sometimes questions about natively 508 00:31:00,310 --> 00:31:03,010 unfolded proteins come up and those 509 00:31:03,010 --> 00:31:05,350 are outside of the scope of our discussions today. 510 00:31:05,350 --> 00:31:08,860 But be aware, there are proteins that are natively unfolded. 511 00:31:08,860 --> 00:31:10,840 You saw a little bit of that with some 512 00:31:10,840 --> 00:31:14,140 of the ribosome proteins that had those unfolded extensions 513 00:31:14,140 --> 00:31:17,320 going into the interior. 514 00:31:17,320 --> 00:31:20,800 So in terms of thinking about protein folding 515 00:31:20,800 --> 00:31:23,730 in the test tube, where we're going to begin 516 00:31:23,730 --> 00:31:28,750 is with Anfinsen's hypothesis and his seminal experiment 517 00:31:28,750 --> 00:31:29,770 on protein folding. 518 00:31:38,270 --> 00:31:44,440 So Anfinsen is responsible for the thermodynamic hypothesis 519 00:31:44,440 --> 00:31:46,420 of protein folding. 520 00:31:46,420 --> 00:31:48,700 And he performed seminal experiments 521 00:31:48,700 --> 00:31:54,170 on a protein, an enzyme, called ribonuclease A. 522 00:31:54,170 --> 00:31:57,820 And so what Anfinsen hypothesized 523 00:31:57,820 --> 00:32:01,660 is that, in terms of a protein shape or fold, 524 00:32:01,660 --> 00:32:05,590 it's the primary sequence, so the sequence of amino acids, 525 00:32:05,590 --> 00:32:10,300 that dictates this final shape in aqueous solution. 526 00:32:10,300 --> 00:32:12,640 So whatever that primary sequence is, 527 00:32:12,640 --> 00:32:14,710 it dictates, basically, the array 528 00:32:14,710 --> 00:32:17,980 of possibilities and the thermodynamically most 529 00:32:17,980 --> 00:32:19,910 favorable result. 530 00:32:19,910 --> 00:32:24,370 So what was the experiment Anfinsen did to probe this? 531 00:32:36,770 --> 00:32:49,770 What he did is look at denaturation and refolding 532 00:32:49,770 --> 00:33:02,840 of ribonuclease A. So this enzyme 533 00:33:02,840 --> 00:33:06,365 cleaves RNA single stranded. 534 00:33:09,410 --> 00:33:13,640 It's 124 amino acids in length. 535 00:33:13,640 --> 00:33:17,705 And in the native form, it contains four disulfide bonds. 536 00:33:22,240 --> 00:33:24,440 And since there's four disulfide bonds, 537 00:33:24,440 --> 00:33:27,320 there's eight cysteines in the primary sequence. 538 00:33:27,320 --> 00:33:29,600 So two cysteine side chains can come together 539 00:33:29,600 --> 00:33:30,920 to form a disulfide. 540 00:33:52,630 --> 00:33:56,000 And so if we think about eight cysteines 541 00:33:56,000 --> 00:33:58,970 forming four disulfide bonds, there's 542 00:33:58,970 --> 00:34:02,960 many possibilities, in terms of how those cysteines are matched 543 00:34:02,960 --> 00:34:03,770 and the linkages. 544 00:34:03,770 --> 00:34:08,929 So different regioisomers, over 100 possible combinations 545 00:34:08,929 --> 00:34:11,420 of these eight cysteines to get four disulfides. 546 00:34:23,460 --> 00:34:31,380 And only one regioisomer, so one of these combinations, 547 00:34:31,380 --> 00:34:32,429 is the native form. 548 00:34:40,429 --> 00:34:42,409 So one out of over 100. 549 00:34:42,409 --> 00:34:45,020 So these native disulfide linkages 550 00:34:45,020 --> 00:34:49,699 that are formed indigenously are required for activity. 551 00:34:49,699 --> 00:34:54,420 So what was Anfinsen's experiment? 552 00:34:54,420 --> 00:35:00,523 The experiment he did was to take native ribonuclease A, 553 00:35:00,523 --> 00:35:01,940 and I'm going to just sketch that. 554 00:35:08,880 --> 00:35:10,925 So imagine we have the four disulfides. 555 00:35:29,740 --> 00:35:38,410 So first, step 1, he reduced it. 556 00:35:38,410 --> 00:35:42,490 So he added a reducing agent to reduce these disulfides. 557 00:35:42,490 --> 00:35:46,310 We'll talk a little bit more what that might be in a minute. 558 00:35:46,310 --> 00:35:59,320 And so the end result is, rather than having these disulfides, 559 00:35:59,320 --> 00:36:02,200 we have eight free cysteines. 560 00:36:02,200 --> 00:36:11,450 So free meaning not in a disulfide, indicated by SH. 561 00:36:11,450 --> 00:36:12,530 OK, so this is reduced. 562 00:36:22,670 --> 00:36:24,860 And so over the course of this, Anfinsen 563 00:36:24,860 --> 00:36:29,510 developed some assays to monitor for activity of this enzyme. 564 00:36:29,510 --> 00:36:33,200 And what was found is that there is a loss of activity. 565 00:36:33,200 --> 00:36:39,455 Next step, add a denaturant. 566 00:36:43,190 --> 00:36:45,950 So a denaturant is some chemical, 567 00:36:45,950 --> 00:36:49,280 like urea or guanidinium, that is 568 00:36:49,280 --> 00:36:52,830 going to disrupt the fold of the protein. 569 00:36:52,830 --> 00:36:56,690 And in this case, he used urea. 570 00:36:56,690 --> 00:37:00,170 So as this is sketched, this is still folded, 571 00:37:00,170 --> 00:37:02,340 but the disulfides are gone. 572 00:37:02,340 --> 00:37:04,700 OK, so what's the result here? 573 00:37:04,700 --> 00:37:15,620 We get some unfolded polypeptide with the cysteine somewhere. 574 00:37:20,490 --> 00:37:35,230 So this is denatured, so we have no disulfides, no native fold, 575 00:37:35,230 --> 00:37:38,320 and inactive. 576 00:37:38,320 --> 00:37:44,750 It can't cleave the single-stranded RNA. 577 00:37:44,750 --> 00:37:48,220 OK, so we've succeeded in destroying activity 578 00:37:48,220 --> 00:37:50,680 and destroying the fold of this protein. 579 00:37:50,680 --> 00:37:52,610 What did he do next? 580 00:37:52,610 --> 00:37:55,690 So the next step in this experiment 581 00:37:55,690 --> 00:38:00,040 was to ask, OK, if we start with this unfolded polypeptide 582 00:38:00,040 --> 00:38:03,820 that's completely denatured and there's no disulfides, 583 00:38:03,820 --> 00:38:07,990 can it return to this native form 584 00:38:07,990 --> 00:38:14,140 by removal of the denaturant, and then 585 00:38:14,140 --> 00:38:16,000 allowing it to oxidize? 586 00:38:16,000 --> 00:38:18,250 So imagine here, we work backwards. 587 00:38:18,250 --> 00:38:33,540 And step 3, remove the denaturant. 588 00:38:33,540 --> 00:38:37,170 So how that might be done, dialysis 589 00:38:37,170 --> 00:38:40,650 is a way to dialyze away the denaturant. 590 00:38:40,650 --> 00:38:44,490 And then what happens if we allow this to oxidize? 591 00:38:48,090 --> 00:38:52,770 So for instance, air oxidation. 592 00:38:58,720 --> 00:39:02,480 So what he found is that in this order of steps, 593 00:39:02,480 --> 00:39:05,590 so the denaturant's removed and then 594 00:39:05,590 --> 00:39:09,850 the protein is allowed to oxidize, that greater than 90% 595 00:39:09,850 --> 00:39:11,740 of the enzymatic activity was restored. 596 00:39:14,290 --> 00:39:18,100 So you have this denatured polypeptide 597 00:39:18,100 --> 00:39:21,070 and dilute aqueous solution, and work your way back 598 00:39:21,070 --> 00:39:23,480 and you can restore this activity. 599 00:39:23,480 --> 00:39:25,575 You have a question? 600 00:39:25,575 --> 00:39:26,950 AUDIENCE: The intermediate stuff, 601 00:39:26,950 --> 00:39:30,490 where it's reduced but not yet denatured, how do you 602 00:39:30,490 --> 00:39:35,050 confirm that the native fold is still the same or similar? 603 00:39:35,050 --> 00:39:38,080 And what were the results, in terms of activity, 604 00:39:38,080 --> 00:39:39,430 for that intermediate? 605 00:39:39,430 --> 00:39:40,347 ELIZABETH NOLAN: Yeah. 606 00:39:40,347 --> 00:39:43,880 So how could we confirm if the fold is perturbed? 607 00:39:43,880 --> 00:39:46,582 What might be a method to do that? 608 00:39:46,582 --> 00:39:47,790 AUDIENCE: Circular dichroism. 609 00:39:47,790 --> 00:39:49,860 ELIZABETH NOLAN: Yeah, circular dichroism. 610 00:39:49,860 --> 00:39:51,450 So that's one possibility. 611 00:39:51,450 --> 00:39:52,620 Did he have that available? 612 00:39:52,620 --> 00:39:54,630 That's another question, but that 613 00:39:54,630 --> 00:39:58,200 will give you a readout on alpha helix C or beta sheet. 614 00:39:58,200 --> 00:40:00,300 That's one possibility. 615 00:40:00,300 --> 00:40:02,100 You can imagine other possibilities. 616 00:40:02,100 --> 00:40:06,480 Maybe it would run differently on some form of column 617 00:40:06,480 --> 00:40:09,240 there, as a possibility. 618 00:40:09,240 --> 00:40:11,100 There was a loss of activity here. 619 00:40:16,440 --> 00:40:20,190 Was it 100% or less than 100%? 620 00:40:20,190 --> 00:40:21,860 I'm not sure about that detail. 621 00:40:21,860 --> 00:40:22,737 Joanne? 622 00:40:22,737 --> 00:40:23,612 JOANNE: I don't know. 623 00:40:23,612 --> 00:40:27,268 Are you sure they didn't denature before they reduced? 624 00:40:27,268 --> 00:40:29,520 ELIZABETH NOLAN: I think he's done it other ways. 625 00:40:29,520 --> 00:40:32,490 JOANNE: I mean, the protein with a lot of disulfides in it, 626 00:40:32,490 --> 00:40:35,610 they may not be accessible to reductant. 627 00:40:35,610 --> 00:40:38,800 ELIZABETH NOLAN: I mean, often, you add them together 628 00:40:38,800 --> 00:40:41,210 to get here, right? 629 00:40:41,210 --> 00:40:46,210 And he did a lot of experiments, as well, with additives. 630 00:40:46,210 --> 00:40:48,210 And then definitely, in this direction, 631 00:40:48,210 --> 00:40:51,850 my understanding is he performed this both ways. 632 00:40:51,850 --> 00:40:56,250 So effectively, if the denaturant's removed first 633 00:40:56,250 --> 00:41:01,630 and then it oxidizes, versus oxidizing it and then removing 634 00:41:01,630 --> 00:41:04,980 the denaturant, and when it was that later scenario, 635 00:41:04,980 --> 00:41:08,130 that the disulfides were allowed to form first, 636 00:41:08,130 --> 00:41:12,990 the end result was a sample that had negligible activity, less 637 00:41:12,990 --> 00:41:15,450 than 1%. 638 00:41:15,450 --> 00:41:18,450 And so from that you can imagine why 639 00:41:18,450 --> 00:41:23,580 because if this was allowed to oxidize, 640 00:41:23,580 --> 00:41:28,470 it's not pre-folded to allow the correct disulfides to form 641 00:41:28,470 --> 00:41:29,493 here. 642 00:41:29,493 --> 00:41:30,910 AUDIENCE: So with those two steps, 643 00:41:30,910 --> 00:41:32,493 the polypeptide and the karyote enzyme 644 00:41:32,493 --> 00:41:34,750 just folded back up into its original state? 645 00:41:34,750 --> 00:41:36,750 ELIZABETH NOLAN: Yeah, so isn't that incredible? 646 00:41:36,750 --> 00:41:37,375 AUDIENCE: Yeah. 647 00:41:37,375 --> 00:41:39,435 How long did it take? 648 00:41:39,435 --> 00:41:41,310 ELIZABETH NOLAN: For this case, I don't know. 649 00:41:41,310 --> 00:41:43,230 And depending on the polypeptide, 650 00:41:43,230 --> 00:41:47,850 it can vary from a short period of time. 651 00:41:47,850 --> 00:41:50,700 We have examples in my lab, where maybe in 30 minutes, 652 00:41:50,700 --> 00:41:53,250 you can see the properly folded form today, 653 00:41:53,250 --> 00:41:55,650 or even faster than that, depending. 654 00:41:55,650 --> 00:41:59,950 Like, seconds to days, depending on the protein and the size. 655 00:41:59,950 --> 00:42:01,950 Yeah, but that's what's really incredible 656 00:42:01,950 --> 00:42:05,010 about this experiment, just beyond the details 657 00:42:05,010 --> 00:42:06,300 of the ordering. 658 00:42:06,300 --> 00:42:09,990 And what happened is the fact that he could take this 124 659 00:42:09,990 --> 00:42:16,160 residue polypeptide that needs to have four specific 660 00:42:16,160 --> 00:42:19,830 disulfides, and just in dilute aqueous solution-- 661 00:42:19,830 --> 00:42:23,940 without any help, minus here-- 662 00:42:23,940 --> 00:42:26,730 it could come to its native fold. 663 00:42:26,730 --> 00:42:29,220 So this was support of this hypothesis, 664 00:42:29,220 --> 00:42:33,180 that the primary sequence of a polypeptide that can dictate 665 00:42:33,180 --> 00:42:34,590 shape. 666 00:42:34,590 --> 00:42:36,660 And if these polypeptides are allowed 667 00:42:36,660 --> 00:42:38,880 to fold under dilute conditions, where 668 00:42:38,880 --> 00:42:42,390 intermolecular molecular interactions aren't a problem, 669 00:42:42,390 --> 00:42:45,690 they can achieve the thermodynamically most 670 00:42:45,690 --> 00:42:51,450 favorable result. And he did plenty 671 00:42:51,450 --> 00:42:53,910 of additional experiments, too, in terms 672 00:42:53,910 --> 00:42:56,580 of putting additives in and asking, 673 00:42:56,580 --> 00:42:59,415 how do these perturb the results? 674 00:43:03,360 --> 00:43:07,980 So what did he actually have to say from his experiment? 675 00:43:07,980 --> 00:43:11,610 In his words, "the results suggest 676 00:43:11,610 --> 00:43:14,900 that the native molecule is the most stable configuration, 677 00:43:14,900 --> 00:43:18,120 thermodynamically speaking, and the major force 678 00:43:18,120 --> 00:43:21,060 in the correct pairing of sulfhydryl groups and disulfide 679 00:43:21,060 --> 00:43:23,640 linkages is the concerted interaction 680 00:43:23,640 --> 00:43:26,440 of psi chain functional groups distributed 681 00:43:26,440 --> 00:43:28,680 along the primary sequence." 682 00:43:28,680 --> 00:43:30,570 So this primary sequence dictates 683 00:43:30,570 --> 00:43:33,310 the array of possibilities. 684 00:43:33,310 --> 00:43:35,970 So in thinking about that, that brings us 685 00:43:35,970 --> 00:43:40,170 to the paradox of Levinthal here. 686 00:43:40,170 --> 00:43:43,570 So he was thinking about this problem of protein folding 687 00:43:43,570 --> 00:43:48,090 and just thought, well, imagine we have one polypeptide 688 00:43:48,090 --> 00:43:49,870 with 100 amino acids. 689 00:43:49,870 --> 00:43:54,810 So smaller than ribonuclease A. What if each amino acid 690 00:43:54,810 --> 00:43:58,552 had only two possible confirmations? 691 00:43:58,552 --> 00:44:00,510 What does that mean, in terms of possibilities? 692 00:44:00,510 --> 00:44:03,570 We have 2 to the 100th. 693 00:44:03,570 --> 00:44:07,710 So if that polypeptide were to sample 694 00:44:07,710 --> 00:44:10,470 every possible confirmation during folding, 695 00:44:10,470 --> 00:44:13,740 taking just a picosecond per transition, 696 00:44:13,740 --> 00:44:17,010 the time required to fold the protein would be what? 697 00:44:17,010 --> 00:44:20,190 And based on his back of the envelope work here, 698 00:44:20,190 --> 00:44:25,560 it would be ridiculous, longer than the time of the universe. 699 00:44:25,560 --> 00:44:27,540 And that tells us that just can't be, 700 00:44:27,540 --> 00:44:31,020 in terms of how we think about this here. 701 00:44:31,020 --> 00:44:35,350 So each amino acid can't adopt its shape independently. 702 00:44:35,350 --> 00:44:39,120 That's just not working on a biological timescale. 703 00:44:39,120 --> 00:44:41,590 So how do we think about this? 704 00:44:41,590 --> 00:44:45,000 We can use energy landscapes here. 705 00:44:45,000 --> 00:44:48,330 So thinking about tumbling through hills and valleys. 706 00:44:48,330 --> 00:44:53,250 And so basically, we can depict protein folding, 707 00:44:53,250 --> 00:44:56,190 and this is an example, say, in a test tube, 708 00:44:56,190 --> 00:45:01,020 where there's some ensemble of starting unfolded, or partially 709 00:45:01,020 --> 00:45:04,740 folded, structures, and these are of higher energy. 710 00:45:04,740 --> 00:45:08,760 And there'll be some sort of stochastic search, 711 00:45:08,760 --> 00:45:13,770 and basically, these forms will give us 712 00:45:13,770 --> 00:45:16,530 ensemble of partially folded structures 713 00:45:16,530 --> 00:45:20,370 and, ultimately, converge to a native structure here that's 714 00:45:20,370 --> 00:45:23,670 of lower energy than that. 715 00:45:23,670 --> 00:45:26,190 So we have an ensemble of many denatured proteins 716 00:45:26,190 --> 00:45:31,020 that needs to make its way to the native form. 717 00:45:31,020 --> 00:45:37,470 And just looking ahead a bit to our discussions of chaperones, 718 00:45:37,470 --> 00:45:40,650 these proteins that assist in folding, 719 00:45:40,650 --> 00:45:44,220 this is another view of an energy landscape, 720 00:45:44,220 --> 00:45:46,540 but it's taking chaperones into account. 721 00:45:46,540 --> 00:45:49,050 So we have energy here. 722 00:45:49,050 --> 00:45:52,460 And this depiction is from the assigned reading. 723 00:45:52,460 --> 00:45:54,510 It basically divides things up in terms 724 00:45:54,510 --> 00:45:59,340 of productive intramolecular contacts versus intermolecular 725 00:45:59,340 --> 00:46:05,610 contacts that lead to situations like oligomers and aggregates 726 00:46:05,610 --> 00:46:07,230 and fibrils. 727 00:46:07,230 --> 00:46:10,950 So up here at high energy, we have unfolded, or partially 728 00:46:10,950 --> 00:46:12,540 folded, species. 729 00:46:12,540 --> 00:46:14,790 And what we see here-- bless you-- 730 00:46:14,790 --> 00:46:19,320 is that these chaperones are helping 731 00:46:19,320 --> 00:46:21,750 to allow these partially folded states to reach 732 00:46:21,750 --> 00:46:25,170 a native state by helping getting over these barriers. 733 00:46:25,170 --> 00:46:28,620 And the chaperones do not want to have the proteins going 734 00:46:28,620 --> 00:46:33,570 in this direction here to species that are potentially 735 00:46:33,570 --> 00:46:38,040 deleterious and result from intermolecular contact, so 736 00:46:38,040 --> 00:46:40,650 oligomers, fibrils, and aggregates here. 737 00:46:44,190 --> 00:46:47,610 What are some methods, in terms of experimental methods, 738 00:46:47,610 --> 00:46:49,710 for folding? 739 00:46:49,710 --> 00:46:52,170 There are many that can be employed. 740 00:46:52,170 --> 00:46:56,880 So you just need to take studies by a case by case basis. 741 00:46:56,880 --> 00:46:59,610 Commonly used fluorescence, whether that be 742 00:46:59,610 --> 00:47:01,030 native emission from a protein. 743 00:47:01,030 --> 00:47:06,270 So if you imagine, you have, say, a tryptophan. 744 00:47:06,270 --> 00:47:09,660 Emission can vary, depending on where it is in a protein. 745 00:47:09,660 --> 00:47:11,010 Methods like FRET. 746 00:47:11,010 --> 00:47:13,500 We just heard about circular dichroism, which tells us 747 00:47:13,500 --> 00:47:19,200 about secondary structure, NMR, FTIR, stopped-flow, 748 00:47:19,200 --> 00:47:22,470 and there's a large field in computation in theory, 749 00:47:22,470 --> 00:47:25,500 looking at protein folding as well. 750 00:47:25,500 --> 00:47:28,080 What are some methods to denature a protein? 751 00:47:28,080 --> 00:47:30,480 So here, we saw urea used. 752 00:47:30,480 --> 00:47:34,770 There's many others, whether that be heat or pH. 753 00:47:34,770 --> 00:47:37,680 And denatured protein means unfolded protein, 754 00:47:37,680 --> 00:47:42,690 in the context of the lectures in this course. 755 00:47:42,690 --> 00:47:48,120 So often, studies in vitro start from using an unfolded protein 756 00:47:48,120 --> 00:47:52,200 sample, and then you look at how folding progresses. 757 00:47:52,200 --> 00:47:55,230 What are some lessons from in vitro folding studies, 758 00:47:55,230 --> 00:47:57,290 just to keep in mind? 759 00:47:57,290 --> 00:48:01,620 1, every protein's different. 760 00:48:01,620 --> 00:48:05,200 And even proteins that seem similar are very different. 761 00:48:05,200 --> 00:48:07,710 So maybe they have the same secondary or tertiary 762 00:48:07,710 --> 00:48:12,870 structure, some small peptide, but when you try to fold them, 763 00:48:12,870 --> 00:48:15,630 they may require different conditions. 764 00:48:15,630 --> 00:48:17,910 There are multi-dimensional energy landscapes, 765 00:48:17,910 --> 00:48:20,970 like what we saw on the prior slides. 766 00:48:20,970 --> 00:48:26,600 You can often see intermediates along the folding process. 767 00:48:26,600 --> 00:48:30,750 And in dilute aqueous solution, as Anfinsen hypothesized, 768 00:48:30,750 --> 00:48:33,810 primary sequence dictates fold. 769 00:48:33,810 --> 00:48:37,030 Just a note to anyone doing experimental work, 770 00:48:37,030 --> 00:48:41,340 why do we like to use the ice bucket in the cold room 771 00:48:41,340 --> 00:48:44,250 when working with proteins and enzymes? 772 00:48:44,250 --> 00:48:46,530 Many native proteins are only marginally 773 00:48:46,530 --> 00:48:50,070 stable under physiological conditions. 774 00:48:50,070 --> 00:48:55,020 So we can think about a delta G of denaturation per amino acid. 775 00:48:55,020 --> 00:48:59,863 So what this means is use your ice bucket in the cold room 776 00:48:59,863 --> 00:49:01,155 when working with your samples. 777 00:49:03,860 --> 00:49:07,160 Just closing, thinking about protein folding 778 00:49:07,160 --> 00:49:08,720 in vitro versus in vivo. 779 00:49:12,260 --> 00:49:14,660 Do studies in vitro really enhance 780 00:49:14,660 --> 00:49:17,930 our understanding of what's happening in the cell? 781 00:49:17,930 --> 00:49:20,870 Just some observations to keep in mind. 782 00:49:20,870 --> 00:49:23,600 So on the benchtop, folding can occur over 783 00:49:23,600 --> 00:49:26,030 a tremendous timescale. 784 00:49:26,030 --> 00:49:28,360 From nanoseconds to hours, I have here. 785 00:49:28,360 --> 00:49:31,900 It can be days, depending on your peptide and conditions 786 00:49:31,900 --> 00:49:33,410 here. 787 00:49:33,410 --> 00:49:38,000 The studies are generally performed in dilute buffer 788 00:49:38,000 --> 00:49:41,520 and in the absence of any additional protein. 789 00:49:41,520 --> 00:49:43,790 So you have some pure polypeptide 790 00:49:43,790 --> 00:49:47,490 that you want to fold or study and that's what you work with. 791 00:49:47,490 --> 00:49:49,670 And it's found that small proteins will often 792 00:49:49,670 --> 00:49:52,070 fold without assistance here. 793 00:49:52,070 --> 00:49:55,160 So they don't need helpers. 794 00:49:55,160 --> 00:49:58,700 In the cell, how do we think about the rate of folding? 795 00:49:58,700 --> 00:50:01,760 So from one point of view, the rate of folding 796 00:50:01,760 --> 00:50:05,090 is limited by the rate of polypeptide biosynthesis 797 00:50:05,090 --> 00:50:08,710 and how quickly that polypeptide is emerging from the ribosome, 798 00:50:08,710 --> 00:50:11,300 if you're thinking about a nascent chain. 799 00:50:11,300 --> 00:50:13,520 And we can think about the concentration of peptide 800 00:50:13,520 --> 00:50:15,800 coming off the ribosome, which is often 801 00:50:15,800 --> 00:50:18,260 quoted as low micromolar. 802 00:50:18,260 --> 00:50:20,270 And as I mentioned earlier, we really 803 00:50:20,270 --> 00:50:23,360 need to keep in mind that this cellular environment is 804 00:50:23,360 --> 00:50:29,210 very crowded with many different biomolecules and players. 805 00:50:29,210 --> 00:50:32,420 And as a consequence of this crowding, 806 00:50:32,420 --> 00:50:36,020 there's many proteins that help in folding, especially 807 00:50:36,020 --> 00:50:36,950 the chaperones. 808 00:50:36,950 --> 00:50:39,080 We'll see that a number of these chaperones 809 00:50:39,080 --> 00:50:43,340 protect the polypeptide that needs to be folded 810 00:50:43,340 --> 00:50:46,280 from this environment here. 811 00:50:46,280 --> 00:50:49,520 So a take home is that just spontaneous protein folding 812 00:50:49,520 --> 00:50:53,960 in the cell is error prone, if it were to happen, 813 00:50:53,960 --> 00:50:56,300 and that inter and intramolecular 814 00:50:56,300 --> 00:50:58,670 interactions are a big issue. 815 00:50:58,670 --> 00:51:02,900 And so these chaperones are available to help 816 00:51:02,900 --> 00:51:05,540 overcome these issues here. 817 00:51:05,540 --> 00:51:09,080 So where we'll begin on Monday is 818 00:51:09,080 --> 00:51:14,150 looking at trigger factor, GroEL and GroES and DnaK/J 819 00:51:14,150 --> 00:51:15,270 as an overview. 820 00:51:15,270 --> 00:51:18,950 And then we'll work our way through these different systems 821 00:51:18,950 --> 00:51:23,130 for protein folding in the cytoplasm. 822 00:51:23,130 --> 00:51:26,180 [SIDE CONVERSATIONS]