1 00:00:00,000 --> 00:00:02,810 NARRATOR: The following content is provided under a Creative 2 00:00:02,810 --> 00:00:04,380 Commons license. 3 00:00:04,380 --> 00:00:06,670 Your support will help MIT Open Courseware 4 00:00:06,670 --> 00:00:11,010 continue to offer high quality educational resources for free. 5 00:00:11,010 --> 00:00:13,670 To make a donation or view additional materials 6 00:00:13,670 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseware 7 00:00:17,600 --> 00:00:18,800 at ocw.MIT.edu. 8 00:00:27,152 --> 00:00:30,000 PROFESSOR: So we have the five-step model. 9 00:00:30,000 --> 00:00:31,742 And what we're going to do-- 10 00:00:31,742 --> 00:00:33,200 this model was presented last time. 11 00:00:33,200 --> 00:00:35,150 And what we'll do is look at experiments 12 00:00:35,150 --> 00:00:38,990 that were designed to look at the denaturation, 13 00:00:38,990 --> 00:00:43,520 translocation, and degradation processes here. 14 00:00:43,520 --> 00:00:47,510 So one question is, can we separate denaturation 15 00:00:47,510 --> 00:00:50,660 from translocation in experiments 16 00:00:50,660 --> 00:00:53,330 to learn about the rates of each process. 17 00:00:53,330 --> 00:00:56,845 And also, how can we examine the role of ATP? 18 00:00:56,845 --> 00:00:58,720 Because that's a question key question here-- 19 00:00:58,720 --> 00:01:03,920 how is ATP hydrolysis by ClpXP allowing this macromolecular 20 00:01:03,920 --> 00:01:06,830 machine to work? 21 00:01:06,830 --> 00:01:13,310 And so we're going to begin with some experiments that 22 00:01:13,310 --> 00:01:14,960 involve a GFP substrate. 23 00:01:19,620 --> 00:01:31,670 So these are some studies of ClpXP activity with a substrate 24 00:01:31,670 --> 00:01:39,080 that has radio-label GFP ssrA. 25 00:01:39,080 --> 00:01:44,060 And so if we think about this substrate here, 26 00:01:44,060 --> 00:01:47,000 we have a radio-label-- 27 00:01:47,000 --> 00:01:49,490 bless you. 28 00:01:49,490 --> 00:01:52,100 We have green fluorescent protein. 29 00:01:52,100 --> 00:01:54,320 And this has a particular fold. 30 00:01:54,320 --> 00:01:56,990 So we have a folded substrate that's fluorescent. 31 00:02:02,110 --> 00:02:05,860 And here we have our tag that will 32 00:02:05,860 --> 00:02:11,140 direct the GFP to the ClpXP degradation machine. 33 00:02:11,140 --> 00:02:16,960 And so this substrate has been used 34 00:02:16,960 --> 00:02:25,910 to look at both degradation and unfolding. 35 00:02:25,910 --> 00:02:28,460 We'll get to the translocation issue 36 00:02:28,460 --> 00:02:32,270 in the second type of substrate we examine today. 37 00:02:32,270 --> 00:02:35,850 And so if we think about degradation, 38 00:02:35,850 --> 00:02:40,250 this is where the radio-label comes in. 39 00:02:40,250 --> 00:02:43,430 And if we think about unfolding, this 40 00:02:43,430 --> 00:02:45,140 is where the fluorescence comes in. 41 00:02:50,270 --> 00:02:52,360 And so what we're going to look is 42 00:02:52,360 --> 00:02:58,450 at degradation and denaturation assays using this substrate. 43 00:02:58,450 --> 00:03:03,190 And so just as a reminder, for anyone not familiar 44 00:03:03,190 --> 00:03:05,260 with green fluorescent protein, I 45 00:03:05,260 --> 00:03:08,380 might just show you the barrel-like structure here 46 00:03:08,380 --> 00:03:10,540 and the chromophores in the interior. 47 00:03:10,540 --> 00:03:12,520 And so in order for GFP to fluoresce, 48 00:03:12,520 --> 00:03:14,900 it needs to have its proper fold. 49 00:03:14,900 --> 00:03:19,660 And if it's denatured, that fluorescence emission is lost. 50 00:03:19,660 --> 00:03:22,550 So let's first look at a degradation assay. 51 00:03:22,550 --> 00:03:24,100 So this is experiment one. 52 00:03:38,290 --> 00:03:39,550 So what is the experiment? 53 00:03:39,550 --> 00:03:43,960 We have GFP here. 54 00:03:43,960 --> 00:03:46,180 And it has this ssrA tag. 55 00:03:49,420 --> 00:03:58,460 And we're going to incubate GFP with ATP and with ClpXP 56 00:03:58,460 --> 00:04:01,220 for some period of time. 57 00:04:01,220 --> 00:04:04,310 And then we're going to stop this reaction with a quench. 58 00:04:04,310 --> 00:04:05,510 And the quench will be acid. 59 00:04:12,710 --> 00:04:16,490 And so if we think about this protein and this degradation 60 00:04:16,490 --> 00:04:19,910 process by ClpXP, what are possible products? 61 00:04:19,910 --> 00:04:27,920 So maybe there's some GFP ssrA that hasn't yet been degraded, 62 00:04:27,920 --> 00:04:30,440 depending on your time point. 63 00:04:30,440 --> 00:04:35,440 And we can imagine some of these short polypeptide fragments 64 00:04:35,440 --> 00:04:38,540 of seven to eight amino acids. 65 00:04:38,540 --> 00:04:41,920 And so we have this radio-label. 66 00:04:41,920 --> 00:04:44,960 And what we want to do is track the radio-label. 67 00:04:44,960 --> 00:04:49,700 So here, we have radio-labeled protein. 68 00:04:54,870 --> 00:04:58,725 And here, we have radio-labeled peptides. 69 00:05:02,400 --> 00:05:05,460 And so if we want to quantify how much degradation occurred, 70 00:05:05,460 --> 00:05:09,210 somehow, we need to separate these. 71 00:05:09,210 --> 00:05:13,440 And so what is a way we can do that here-- something simple. 72 00:05:13,440 --> 00:05:15,090 And so what you want to think about 73 00:05:15,090 --> 00:05:19,810 is just relative solubility under acidic conditions. 74 00:05:19,810 --> 00:05:22,350 So if we have this large GFP that's 75 00:05:22,350 --> 00:05:25,920 folded, when that is treated with acid, 76 00:05:25,920 --> 00:05:28,830 GFP is going to precipitate. 77 00:05:28,830 --> 00:05:30,300 So this will be insoluble. 78 00:05:35,680 --> 00:05:38,460 And in contrast, these peptide fragments 79 00:05:38,460 --> 00:05:40,560 will be soluble, in most instances. 80 00:05:45,410 --> 00:05:47,570 So as a result, we can take advantage 81 00:05:47,570 --> 00:05:51,020 of this differing solubility, effectively 82 00:05:51,020 --> 00:05:52,145 to centrifuge the mixture. 83 00:05:59,260 --> 00:06:08,560 And we can measure the radioactivity in the pellet 84 00:06:08,560 --> 00:06:09,853 and in the supernatant. 85 00:06:15,050 --> 00:06:24,280 And then we can quantify degradation here. 86 00:06:28,550 --> 00:06:30,250 And so what are the results? 87 00:06:34,810 --> 00:06:42,610 So the result-- here, we can imagine a plot 88 00:06:42,610 --> 00:06:54,350 where we have percent of the substrate versus time. 89 00:06:57,840 --> 00:07:01,460 And these were conducted over the course of an hour. 90 00:07:05,240 --> 00:07:08,540 And what's observed here-- 91 00:07:08,540 --> 00:07:12,410 for instance, we have reactions where the substrate was 92 00:07:12,410 --> 00:07:17,240 incubated with ClpXP and ATP. 93 00:07:17,240 --> 00:07:19,700 So we see that over time, there's 94 00:07:19,700 --> 00:07:25,010 a decrease in the percent of GFP ssrA. 95 00:07:25,010 --> 00:07:28,850 And from these data, we can get some degradation rate. 96 00:07:28,850 --> 00:07:30,590 And we'll come back to that degradation 97 00:07:30,590 --> 00:07:32,840 rate in a little bit here. 98 00:07:32,840 --> 00:07:36,650 So what we see here is degradation. 99 00:07:41,050 --> 00:07:45,400 What happens if we add an inhibitor of the protease? 100 00:07:45,400 --> 00:07:47,230 So in the introductory lecture, we 101 00:07:47,230 --> 00:07:50,960 talked about a number of different types of inhibitors. 102 00:07:50,960 --> 00:07:53,600 And so that experiment was done. 103 00:07:53,600 --> 00:08:07,150 And so here, if we take ClpXP plus ATP plus inhibitor, 104 00:08:07,150 --> 00:08:09,160 what we see is no degradation. 105 00:08:19,230 --> 00:08:22,660 And the name of this inhibitor is DFP. 106 00:08:22,660 --> 00:08:25,880 And effectively, it covalently modifies the serine, 107 00:08:25,880 --> 00:08:29,130 in terms of what was used. 108 00:08:29,130 --> 00:08:33,289 So what do we conclude from these data? 109 00:08:33,289 --> 00:08:36,409 If the active site serine of ClpX 110 00:08:36,409 --> 00:08:39,470 is covalently modified with an inhibitor, which 111 00:08:39,470 --> 00:08:44,090 is diisopropyl fluorophosphate, we lose activity. 112 00:08:44,090 --> 00:08:48,080 So that serine is important. 113 00:08:48,080 --> 00:08:55,130 So what about unfolding or denaturation? 114 00:08:55,130 --> 00:08:56,990 How can we get at that? 115 00:08:56,990 --> 00:08:58,520 So that will be experiment two. 116 00:09:02,630 --> 00:09:11,950 And in terms of thinking about denaturation, 117 00:09:11,950 --> 00:09:13,450 rather than the radio-label, we're 118 00:09:13,450 --> 00:09:16,750 going to think about the GFP. 119 00:09:16,750 --> 00:09:22,770 And so imagine we have our folded GFP-- 120 00:09:22,770 --> 00:09:24,820 however we want to show that here-- 121 00:09:24,820 --> 00:09:28,930 that has this ssrA tag. 122 00:09:28,930 --> 00:09:33,447 So this is folded and fluorescent. 123 00:09:37,920 --> 00:09:47,410 So this gets denatured by ClpX of the machine 124 00:09:47,410 --> 00:09:51,220 to give us some unfolded polypeptide that 125 00:09:51,220 --> 00:09:54,190 has this ssrA tag-- 126 00:09:54,190 --> 00:09:58,510 unfolded and non-fluorescent. 127 00:10:04,180 --> 00:10:05,713 And then what happens? 128 00:10:05,713 --> 00:10:06,505 This gets degraded. 129 00:10:15,060 --> 00:10:16,590 And we get these fragments. 130 00:10:20,850 --> 00:10:23,280 And these fragments are also non-fluorescent. 131 00:10:28,060 --> 00:10:31,840 So effectively, s we can perform the exact same assay 132 00:10:31,840 --> 00:10:34,270 as we did an experiment one. 133 00:10:34,270 --> 00:10:37,090 But we'll look at fluorescence as a readout 134 00:10:37,090 --> 00:10:39,690 rather than quantifying radioactivity. 135 00:10:39,690 --> 00:10:44,800 STUDENT: So If you take ssrA on any protein, 136 00:10:44,800 --> 00:10:47,770 would ClpXP break it down? 137 00:10:50,410 --> 00:10:52,362 [INAUDIBLE] 138 00:10:52,362 --> 00:10:59,050 PROFESSOR: Yeah, so GFP probably isn't a native substrate of-- 139 00:10:59,050 --> 00:11:00,400 definitely not of E. coli ClpXP. 140 00:11:02,980 --> 00:11:05,650 What happens in a system that expresses GFP natively, 141 00:11:05,650 --> 00:11:07,150 I'm not sure. 142 00:11:07,150 --> 00:11:11,200 But yes, this has been a wonderful tool for experiments 143 00:11:11,200 --> 00:11:14,530 because many different protein substrates 144 00:11:14,530 --> 00:11:19,360 can be modified with this ssrA tag and directed to ClpXP. 145 00:11:19,360 --> 00:11:21,380 This is just one example. 146 00:11:21,380 --> 00:11:23,800 So I think broadly, we can think that there 147 00:11:23,800 --> 00:11:27,100 are many, many possibilities for what can be delivered. 148 00:11:27,100 --> 00:11:31,890 Are there certain proteins that ClpXP just can't deal with? 149 00:11:31,890 --> 00:11:34,330 That's a possibility. 150 00:11:34,330 --> 00:11:36,970 So the problem set for the upcoming week 151 00:11:36,970 --> 00:11:39,400 has a case where there's a disulfide bond, for instance, 152 00:11:39,400 --> 00:11:42,520 and asking what happens when we have 153 00:11:42,520 --> 00:11:44,230 some other types of structural features 154 00:11:44,230 --> 00:11:47,710 within a designed substrate. 155 00:11:47,710 --> 00:11:49,450 But for the purposes of this, yes, we 156 00:11:49,450 --> 00:11:52,990 can attach ssrA on to some protein 157 00:11:52,990 --> 00:11:55,720 that we can use to study the system 158 00:11:55,720 --> 00:11:57,460 and therefore do the experiments. 159 00:12:01,250 --> 00:12:03,670 And does that make sense, also, just 160 00:12:03,670 --> 00:12:07,480 thinking from the standpoint of what types of polypeptides 161 00:12:07,480 --> 00:12:10,720 might get directed to ClpXP in vivo? 162 00:12:10,720 --> 00:12:15,340 The ribosome could stall with many different types 163 00:12:15,340 --> 00:12:17,890 of proteins being synthesized there. 164 00:12:17,890 --> 00:12:20,980 So pretty versatile here. 165 00:12:20,980 --> 00:12:24,280 So we're going to perform the same assay. 166 00:12:24,280 --> 00:12:26,890 But we're going to measure fluorescence 167 00:12:26,890 --> 00:12:28,960 rather than radioactivity. 168 00:12:28,960 --> 00:12:30,580 And so what is the result? 169 00:12:35,930 --> 00:12:53,220 So here, we have fluorescence. 170 00:12:53,220 --> 00:12:56,325 And again, now, we have the percent of folded GFP-- 171 00:13:00,200 --> 00:13:02,660 100. 172 00:13:02,660 --> 00:13:06,030 And again, we can we can imagine this going down zero 173 00:13:06,030 --> 00:13:06,870 to 60 minutes. 174 00:13:13,420 --> 00:13:14,500 So here we have ClpXP. 175 00:13:20,310 --> 00:13:24,660 What happens if we have the inhibitor, for instance? 176 00:13:24,660 --> 00:13:36,920 What they found-- and I'll draw the inhibitor in a minute 177 00:13:36,920 --> 00:13:39,800 because I'm sure some of you are wondering. 178 00:13:39,800 --> 00:13:45,140 And here we have ClpX alone. 179 00:13:45,140 --> 00:13:52,370 So how do we interpret these data? 180 00:13:52,370 --> 00:13:55,160 So if we have the full machinery-- 181 00:13:55,160 --> 00:13:59,720 ClpXP and ATP-- we see a loss in fluorescence 182 00:13:59,720 --> 00:14:03,680 over time, which indicates a loss in folded GFP. 183 00:14:03,680 --> 00:14:06,620 So the substrate is being denatured. 184 00:14:06,620 --> 00:14:09,680 What about this case here when we only have ClpX present? 185 00:14:12,630 --> 00:14:13,950 And also, it won't have ATP. 186 00:14:17,110 --> 00:14:18,190 What's happening there? 187 00:14:27,185 --> 00:14:31,560 STUDENT: Without ClpP, there's no actual degradation 188 00:14:31,560 --> 00:14:33,030 that goes on. 189 00:14:33,030 --> 00:14:35,920 PROFESSOR: Do we need to see degradation in this assay? 190 00:14:35,920 --> 00:14:40,510 That's true, but what is this assay giving us a readout on? 191 00:14:40,510 --> 00:14:42,580 Just unfolding. 192 00:14:42,580 --> 00:14:44,790 So what do we learn from that? 193 00:14:44,790 --> 00:14:45,290 Rebecca? 194 00:14:45,290 --> 00:14:47,790 STUDENT: ClpX needs to be found to ClpP 195 00:14:47,790 --> 00:14:51,612 to be in the correct confirmation to unfold. 196 00:14:51,612 --> 00:14:52,900 PROFESSOR: Yes, yes. 197 00:14:52,900 --> 00:14:55,150 So this indicates that ClpX and ClpP 198 00:14:55,150 --> 00:14:59,590 need to be in complex in order to allow unfolding to occur. 199 00:14:59,590 --> 00:15:01,480 So thinking to the cellular environment, 200 00:15:01,480 --> 00:15:02,330 does not make sense? 201 00:15:06,838 --> 00:15:08,130 Yeah, I'm seeing nodding heads. 202 00:15:08,130 --> 00:15:09,750 Yes, right. 203 00:15:09,750 --> 00:15:12,450 So we wouldn't want just ClpX to be able to bind 204 00:15:12,450 --> 00:15:18,360 and unfold anything it comes into contact with there. 205 00:15:18,360 --> 00:15:20,370 And in terms of this inhibitor, we're 206 00:15:20,370 --> 00:15:22,740 seeing that it's not unfolding very well. 207 00:15:22,740 --> 00:15:24,720 So this inhibitor is for the protease. 208 00:15:24,720 --> 00:15:31,980 Just for that structure, effectively, 209 00:15:31,980 --> 00:15:35,380 what we have here-- 210 00:15:41,270 --> 00:15:51,270 you actually saw this in the lecture slides from last time. 211 00:15:51,270 --> 00:15:54,110 So this is DFP. 212 00:15:54,110 --> 00:15:58,170 And effectively, what it does is it 213 00:15:58,170 --> 00:16:05,190 will modify a serine side chain to give us this species here. 214 00:16:05,190 --> 00:16:07,260 And that will block proteolytic activity. 215 00:16:13,110 --> 00:16:15,120 So how did these data compare? 216 00:16:18,700 --> 00:16:22,860 How does the denaturation and degradation data compare? 217 00:16:22,860 --> 00:16:26,250 And so we can look at what was done. 218 00:16:26,250 --> 00:16:29,380 And effectively, what we want to ask 219 00:16:29,380 --> 00:16:34,600 is, how did the steady state kinetic data compare? 220 00:16:34,600 --> 00:16:37,390 And so steady state experiments were done, of course, 221 00:16:37,390 --> 00:16:39,250 with varying substrate. 222 00:16:39,250 --> 00:16:41,260 And the data were re-plotted. 223 00:16:41,260 --> 00:16:44,480 And so those data are shown here. 224 00:16:44,480 --> 00:16:47,590 And what we're looking at on the y-axis 225 00:16:47,590 --> 00:16:51,430 is the loss of substrates-- so GFP ssrA 226 00:16:51,430 --> 00:16:55,150 versus the concentration of substrate. 227 00:16:55,150 --> 00:16:57,580 And what we see is that in circles, 228 00:16:57,580 --> 00:16:59,650 we have the fluorescence data. 229 00:16:59,650 --> 00:17:03,920 And in triangles, we have the data from radioactivity. 230 00:17:06,849 --> 00:17:09,040 So what does this analysis tell us? 231 00:17:28,068 --> 00:17:30,110 STUDENT: The data set doesn't look that complete. 232 00:17:30,110 --> 00:17:32,420 But it looks like they're on about the same time scale. 233 00:17:32,420 --> 00:17:34,760 PROFESSOR: They look very similar. 234 00:17:34,760 --> 00:17:38,000 We're getting the same steady state kinetic parameters 235 00:17:38,000 --> 00:17:41,370 for both analyses here. 236 00:17:41,370 --> 00:17:43,280 And yes, it might be nice to have more data. 237 00:17:43,280 --> 00:17:45,560 But that's just not available. 238 00:17:45,560 --> 00:17:48,705 So all of these data can be fit to the same kcat and km. 239 00:17:51,620 --> 00:17:56,570 So what do these data tell us about a rate determining step, 240 00:17:56,570 --> 00:17:58,730 for instance? 241 00:17:58,730 --> 00:17:59,495 Not very much. 242 00:18:02,690 --> 00:18:07,190 And we also haven't yet thought about this issue 243 00:18:07,190 --> 00:18:08,660 of translocation. 244 00:18:08,660 --> 00:18:10,700 We're just seeing the unfolding step 245 00:18:10,700 --> 00:18:14,810 and seeing the degradation step in this assay here. 246 00:18:14,810 --> 00:18:16,570 So we need some more information. 247 00:18:23,240 --> 00:18:25,050 So if we think about this, we have 248 00:18:25,050 --> 00:18:43,470 denaturation versus translocation degradation. 249 00:18:46,640 --> 00:18:50,070 And so far, we've been able to look at this and this. 250 00:18:50,070 --> 00:18:52,140 And our intuition tells us degradation 251 00:18:52,140 --> 00:18:53,790 by the protease should be very fast. 252 00:18:56,310 --> 00:19:00,960 So can we learn something about translocation 253 00:19:00,960 --> 00:19:07,200 which we weren't able to see in these experiments here? 254 00:19:07,200 --> 00:19:09,630 And so that's what we want to focus on now 255 00:19:09,630 --> 00:19:20,420 because there was no readout on this step from experiments one 256 00:19:20,420 --> 00:19:24,560 and two here. 257 00:19:24,560 --> 00:19:27,980 So is it possible to separate denaturation and translocation 258 00:19:27,980 --> 00:19:30,401 with some strategically designed substrates? 259 00:19:35,111 --> 00:19:37,990 STUDENT: From this experiment, can't we 260 00:19:37,990 --> 00:19:40,480 deduce that translocation step is 261 00:19:40,480 --> 00:19:43,380 much slower than denaturation? 262 00:19:43,380 --> 00:19:44,210 PROFESSOR: Can we? 263 00:19:44,210 --> 00:19:44,710 How? 264 00:19:47,810 --> 00:19:50,860 Yeah, there's just no readout because this loss 265 00:19:50,860 --> 00:19:53,920 in fluorescence is just telling us that the protein is folded 266 00:19:53,920 --> 00:19:55,180 or unfolded. 267 00:19:55,180 --> 00:19:56,650 And the degradation is just telling 268 00:19:56,650 --> 00:19:58,990 us what happens in the protease chamber. 269 00:19:58,990 --> 00:20:00,970 So what happens from that point-- 270 00:20:00,970 --> 00:20:07,450 unfolding to degradation-- in between, we don't know here. 271 00:20:07,450 --> 00:20:13,540 So what we need is a new set of substrates 272 00:20:13,540 --> 00:20:18,580 that are going to let us get at this 273 00:20:18,580 --> 00:20:23,050 and allow us to separate denaturation from translocation 274 00:20:23,050 --> 00:20:25,060 experimentally. 275 00:20:25,060 --> 00:20:28,240 And so what was the idea for doing this? 276 00:20:28,240 --> 00:20:33,280 The idea was to take some protein that's been studied 277 00:20:33,280 --> 00:20:36,190 and take that protein and a series of mutants 278 00:20:36,190 --> 00:20:38,830 of that protein that have also been studied. 279 00:20:38,830 --> 00:20:42,670 And the key here is that the mutants of the protein 280 00:20:42,670 --> 00:20:44,770 have varying instabilities-- 281 00:20:44,770 --> 00:20:48,160 so varying instabilities of the fold. 282 00:20:48,160 --> 00:20:51,370 And so you can imagine that there 283 00:20:51,370 --> 00:20:53,320 have been many studies of protein folding 284 00:20:53,320 --> 00:20:56,440 out there asking the consequences of making 285 00:20:56,440 --> 00:21:00,280 point mutations in a given protein fold on stability 286 00:21:00,280 --> 00:21:01,150 there. 287 00:21:01,150 --> 00:21:04,540 And so that's exactly what was done. 288 00:21:04,540 --> 00:21:22,750 So what we need is a new set of substrates to probe effectively 289 00:21:22,750 --> 00:21:40,140 denaturation and translocation in more detail here. 290 00:21:40,140 --> 00:21:46,260 And the key question is, is it possible 291 00:21:46,260 --> 00:21:59,120 to separate denaturation from translocation? 292 00:22:04,740 --> 00:22:20,140 And so what was done is to take an immunoglobulin-like domain 293 00:22:20,140 --> 00:22:22,810 from a protein found in striated muscle that 294 00:22:22,810 --> 00:22:25,990 has been the subject of many studies 295 00:22:25,990 --> 00:22:30,195 and mutants of this protein and to employ them in assays. 296 00:22:36,120 --> 00:22:49,230 So we're going to take a protein plus variants 297 00:22:49,230 --> 00:23:09,130 with varying stabilities and perform this assay 298 00:23:09,130 --> 00:23:10,720 and compare the data. 299 00:23:10,720 --> 00:23:17,920 And so here is the protein that was used as a model substrate. 300 00:23:17,920 --> 00:23:21,880 So shown here, this is the titin I27 domain 301 00:23:21,880 --> 00:23:25,060 that has an ssrA tag attached. 302 00:23:25,060 --> 00:23:28,720 OK so if we take a look at this protein that 303 00:23:28,720 --> 00:23:35,500 has a beta sandwich fold, we see that there's a disulfide bond. 304 00:23:35,500 --> 00:23:37,510 There's a single tryptophan residue. 305 00:23:37,510 --> 00:23:39,970 And this is helpful because tryptophan residues 306 00:23:39,970 --> 00:23:41,610 have intrinsic fluorescence that's 307 00:23:41,610 --> 00:23:44,050 sensitive to the environment. 308 00:23:44,050 --> 00:23:46,720 And we see it's buried in the inside here. 309 00:23:46,720 --> 00:23:49,480 So in a hydrophobic versus hydrophobic environment, 310 00:23:49,480 --> 00:23:51,220 the fluorescence will differ. 311 00:23:51,220 --> 00:23:56,680 And so we can use that as a readout of unfolding here. 312 00:23:56,680 --> 00:24:00,790 And this is just an example of data from a prior study 313 00:24:00,790 --> 00:24:05,380 where this protein and various mutants 314 00:24:05,380 --> 00:24:12,340 of the protein like here, valine 11P Y9P 315 00:24:12,340 --> 00:24:16,100 were studied for stability of the fold. 316 00:24:16,100 --> 00:24:18,190 So guanidinium, we learned that's the denaturant 317 00:24:18,190 --> 00:24:20,230 in the folding section. 318 00:24:20,230 --> 00:24:25,230 So these various point mutations have different stabilities. 319 00:24:25,230 --> 00:24:28,390 And we can see that in these denaturation curves here. 320 00:24:31,100 --> 00:24:35,050 So what was done in their experiments 321 00:24:35,050 --> 00:24:38,020 were very similar to what was done before. 322 00:24:38,020 --> 00:24:45,340 So we take this titin radio-labeled-- 323 00:24:45,340 --> 00:24:45,940 bless you. 324 00:24:45,940 --> 00:24:57,350 So this is experiment three with the ssrA tag. 325 00:25:00,230 --> 00:25:08,840 Incubate with ClpXP with ATP and asks what happened. 326 00:25:08,840 --> 00:25:11,240 And in terms of these substrates, 327 00:25:11,240 --> 00:25:23,300 we have the wild-type, we have the mutants, as shown up here. 328 00:25:23,300 --> 00:25:26,750 And we have CM, you'll see in the data, 329 00:25:26,750 --> 00:25:27,965 which is chemically modified. 330 00:25:35,790 --> 00:25:38,600 And these chemically modified variants 331 00:25:38,600 --> 00:25:40,970 are completely denatured-- we can consider them. 332 00:25:49,480 --> 00:25:52,660 And so effectively, what was done here 333 00:25:52,660 --> 00:26:00,970 with cysteine modification, with iodoacetamide. 334 00:26:04,760 --> 00:26:09,500 So we saw that in discussions-- introductory discussions-- 335 00:26:09,500 --> 00:26:12,290 about unnatural amino acid incorporation. 336 00:26:12,290 --> 00:26:16,140 So the disulfide bond is completely disrupted here. 337 00:26:16,140 --> 00:26:19,170 The disulfide can be reduced, the cysteines modified, 338 00:26:19,170 --> 00:26:22,550 and we get an unfolded version here for that. 339 00:26:26,990 --> 00:26:32,600 And here what do we find? 340 00:26:32,600 --> 00:26:36,440 So there's a number of different point mutants 341 00:26:36,440 --> 00:26:38,820 that are listed here. 342 00:26:38,820 --> 00:26:40,790 And we're just going to look at a few, 343 00:26:40,790 --> 00:26:42,610 in terms of what they found. 344 00:26:45,310 --> 00:26:49,180 So in terms of degradation assay, which 345 00:26:49,180 --> 00:27:11,010 is how they did this readout, we're 346 00:27:11,010 --> 00:27:15,960 going to have the percent titin remaining. 347 00:27:22,860 --> 00:27:28,860 So again, using radioactivity in the supernatant or pellet 348 00:27:28,860 --> 00:27:29,640 versus time-- 349 00:27:45,380 --> 00:27:46,220 what did they find? 350 00:27:46,220 --> 00:27:52,070 So if we take a look at a selection of the data-- 351 00:27:52,070 --> 00:27:54,230 just put three examples-- 352 00:27:57,600 --> 00:27:58,755 here, what do we have? 353 00:28:02,720 --> 00:28:03,950 Here, we have wild-type. 354 00:28:08,710 --> 00:28:10,720 Here, we have one of the mutants, B13P. 355 00:28:13,330 --> 00:28:16,480 And here, we have chemically modified wild-type. 356 00:28:23,350 --> 00:28:26,050 So what do these data tell us? 357 00:28:46,010 --> 00:28:48,006 STUDENT: Degradation is faster. 358 00:28:48,006 --> 00:28:49,004 It's [INAUDIBLE] 359 00:28:49,004 --> 00:28:50,420 PROFESSOR: Yeah. 360 00:28:50,420 --> 00:28:52,460 That's one thing we see here. 361 00:28:52,460 --> 00:28:55,850 So this chemically modified protein is denatured. 362 00:28:55,850 --> 00:28:59,540 And we see that the denatured protein is easier 363 00:28:59,540 --> 00:29:04,370 to degrade by ClpXP than the native protein. 364 00:29:04,370 --> 00:29:07,580 We also see that the mutant is more rapidly 365 00:29:07,580 --> 00:29:09,030 degraded than the wild-type. 366 00:29:09,030 --> 00:29:13,430 So ClpXP is having an easier time with this one here too. 367 00:29:13,430 --> 00:29:15,110 So there's an apparent correlation 368 00:29:15,110 --> 00:29:18,590 here between the ease of unfolding 369 00:29:18,590 --> 00:29:20,690 and the ease of degradation. 370 00:29:20,690 --> 00:29:23,480 A protein that's already unfolded or is relatively 371 00:29:23,480 --> 00:29:26,480 easy to fold is degraded more rapidly 372 00:29:26,480 --> 00:29:28,310 than the wild-type protein that has 373 00:29:28,310 --> 00:29:31,970 this beta sandwich fold here. 374 00:29:31,970 --> 00:29:37,790 If we think about the processes happening in each of these 375 00:29:37,790 --> 00:29:40,340 and we think back to that five-step model, 376 00:29:40,340 --> 00:29:41,790 what's happening? 377 00:29:41,790 --> 00:29:50,270 So here, we have denaturation plus translocation 378 00:29:50,270 --> 00:29:53,480 plus degradation. 379 00:29:53,480 --> 00:29:57,470 And likewise, here, we have these three parameters as well. 380 00:30:04,350 --> 00:30:07,830 And in this case, we don't have denaturation. 381 00:30:07,830 --> 00:30:14,250 We just have the translocation and the degradation here. 382 00:30:23,770 --> 00:30:26,310 STUDENT: Why are the rates linear here? 383 00:30:26,310 --> 00:30:29,640 And it was not linear in the previous one. 384 00:30:29,640 --> 00:30:31,230 PROFESSOR: Just imagine this is-- 385 00:30:31,230 --> 00:30:34,020 well, one is a completely different substrate. 386 00:30:34,020 --> 00:30:36,840 The time frame, I haven't given here. 387 00:30:36,840 --> 00:30:38,460 Don't worry about that. 388 00:30:38,460 --> 00:30:39,980 We're just looking at that one part. 389 00:30:44,850 --> 00:30:49,350 So here's the actual data from the report. 390 00:30:49,350 --> 00:30:54,900 And now, what we want to do is, using this whole family 391 00:30:54,900 --> 00:30:56,280 of substrates-- 392 00:30:56,280 --> 00:31:00,510 so the native I27 domain, the various point mutants, 393 00:31:00,510 --> 00:31:02,640 and these chemically modified forms, 394 00:31:02,640 --> 00:31:05,040 we want to look at the details of the steady state 395 00:31:05,040 --> 00:31:07,830 kinetic analysis. 396 00:31:07,830 --> 00:31:12,300 And we also want to look at what's going on with the ATPs. 397 00:31:12,300 --> 00:31:15,060 So what is the rate of ATP hydrolysis. 398 00:31:15,060 --> 00:31:18,690 And how many ATPs are hydrolyzed? 399 00:31:18,690 --> 00:31:21,570 We know nothing about that yet, in terms of the data that's 400 00:31:21,570 --> 00:31:24,690 been presented so far. 401 00:31:24,690 --> 00:31:28,440 So what we're going to do is take a look at this dataset 402 00:31:28,440 --> 00:31:31,710 and see what we learn here. 403 00:31:31,710 --> 00:31:33,580 So there's quite a bit of data in here. 404 00:31:33,580 --> 00:31:37,140 But we're just going to systematically work through. 405 00:31:37,140 --> 00:31:40,110 So what do we see? 406 00:31:40,110 --> 00:31:43,110 Here, we have all of the different I27 407 00:31:43,110 --> 00:31:46,110 domain-based substrates they used. 408 00:31:46,110 --> 00:31:50,550 And the table is divided basically in terms of 409 00:31:50,550 --> 00:31:53,760 whether or not the protein was chemically modified. 410 00:31:53,760 --> 00:31:56,040 So on top, we have wild type. 411 00:31:56,040 --> 00:31:57,880 And then we have these four-- 412 00:31:57,880 --> 00:31:59,990 or sorry, five-- point mutants. 413 00:31:59,990 --> 00:32:01,710 And in this bottom half of the table, 414 00:32:01,710 --> 00:32:04,260 we have the chemically modified wild type 415 00:32:04,260 --> 00:32:07,710 and the chemically modified point mutants. 416 00:32:07,710 --> 00:32:11,580 So these begin with a fold, and depending on the mutation here, 417 00:32:11,580 --> 00:32:15,240 there's differing stability of that fold. 418 00:32:15,240 --> 00:32:17,160 And here, we have unfolded variants 419 00:32:17,160 --> 00:32:20,370 because the disulfide was disrupted. 420 00:32:20,370 --> 00:32:21,570 So what are we looking at? 421 00:32:21,570 --> 00:32:27,900 We have degradation, we have km, we have denaturation, 422 00:32:27,900 --> 00:32:30,600 and then we have the ATP S rate, and the number 423 00:32:30,600 --> 00:32:36,130 of ATPs per I27 domain degraded here. 424 00:32:36,130 --> 00:32:41,110 So the question is, what do we learn from each column of data? 425 00:32:41,110 --> 00:32:47,950 So if we take a look at these degradation rates here, 426 00:32:47,950 --> 00:32:48,840 what do we see? 427 00:32:54,020 --> 00:32:57,260 So what happens amongst the proteins that 428 00:32:57,260 --> 00:32:58,470 are not chemically modified? 429 00:33:05,280 --> 00:33:07,170 And don't try to over-analyze it, 430 00:33:07,170 --> 00:33:12,620 just look for what are the obvious differences here. 431 00:33:21,078 --> 00:33:21,995 So what's the slowest? 432 00:33:25,510 --> 00:33:26,740 Wild-type, right? 433 00:33:26,740 --> 00:33:29,770 Similar to what we saw here, and that makes sense 434 00:33:29,770 --> 00:33:33,340 because wild-type has the most stable fold, just 435 00:33:33,340 --> 00:33:35,240 based on what we saw here. 436 00:33:35,240 --> 00:33:38,290 And then what do we see for the mutants? 437 00:33:38,290 --> 00:33:41,040 There's variability. 438 00:33:41,040 --> 00:33:44,170 And all of these values are greater. 439 00:33:44,170 --> 00:33:47,440 How do they compare to chemically modified variants? 440 00:33:47,440 --> 00:33:48,760 And what do we see here? 441 00:33:52,110 --> 00:33:54,380 These are the fastest. 442 00:33:54,380 --> 00:33:57,770 And they're all pretty similar. 443 00:33:57,770 --> 00:34:01,130 So these data agree with what we drew up here. 444 00:34:05,180 --> 00:34:06,425 What about the km values? 445 00:34:12,920 --> 00:34:15,590 So are these all similar or different? 446 00:34:19,270 --> 00:34:21,066 All pretty similar, yeah. 447 00:34:21,066 --> 00:34:22,274 And why does that make sense? 448 00:34:25,199 --> 00:34:27,179 So that indicates that ClpX binds 449 00:34:27,179 --> 00:34:30,249 all of these substrates in a similar way. 450 00:34:36,830 --> 00:34:39,448 They all have the ssrA tag there. 451 00:34:43,810 --> 00:34:45,760 So we can't attribute any changes 452 00:34:45,760 --> 00:34:49,989 we're seeing in rate to this km value here. 453 00:34:49,989 --> 00:34:51,714 What about these denaturation rates? 454 00:34:54,840 --> 00:34:59,070 So we don't have any values for the chemically modified forms 455 00:34:59,070 --> 00:35:01,470 because they're already denatured. 456 00:35:01,470 --> 00:35:02,410 What do we see? 457 00:35:02,410 --> 00:35:05,500 We see the wild-type is more difficult to denature-- 458 00:35:05,500 --> 00:35:07,410 so the slower rate-- 459 00:35:07,410 --> 00:35:10,292 than these point mutations here. 460 00:35:10,292 --> 00:35:12,750 And you could imagine if you were the researcher going back 461 00:35:12,750 --> 00:35:14,340 and comparing these data to what's 462 00:35:14,340 --> 00:35:17,760 known about the relative stabilities of each fold 463 00:35:17,760 --> 00:35:20,340 from other data in the literature 464 00:35:20,340 --> 00:35:24,300 from studies like that guanidinium denaturation 465 00:35:24,300 --> 00:35:27,960 on the prior slide here. 466 00:35:27,960 --> 00:35:31,740 So what about the data in these columns? 467 00:35:31,740 --> 00:35:32,420 What do we see? 468 00:35:39,860 --> 00:35:42,260 So here, we're looking at ATPase activity. 469 00:35:54,460 --> 00:35:57,550 STUDENT: In that case, it's slower 470 00:35:57,550 --> 00:36:02,072 and less efficient for wild-type than chemically modified. 471 00:36:02,072 --> 00:36:03,670 PROFESSOR: Yes. 472 00:36:03,670 --> 00:36:04,210 Yes. 473 00:36:04,210 --> 00:36:06,940 That's certainly the case. 474 00:36:06,940 --> 00:36:11,380 So, first, if we look at wild-type, and even 475 00:36:11,380 --> 00:36:13,570 for that matter, these single point variants, 476 00:36:13,570 --> 00:36:16,750 versus these chemically modified forms, 477 00:36:16,750 --> 00:36:24,510 we see that the wild-type has a value of about 150 per minute. 478 00:36:24,510 --> 00:36:26,310 And these are slightly higher. 479 00:36:26,310 --> 00:36:30,600 We see these are on the order of about 600 per minute. 480 00:36:30,600 --> 00:36:33,360 So in a way, these fall into two groups-- 481 00:36:33,360 --> 00:36:36,370 the chemically modified forms defined one group. 482 00:36:36,370 --> 00:36:38,070 And this wild-type and single point 483 00:36:38,070 --> 00:36:42,510 mutants define another group here. 484 00:36:42,510 --> 00:36:47,520 And the wild-type has the slowest ATPase right here. 485 00:36:47,520 --> 00:36:50,790 And then in terms of efficiency, as you mentioned-- 486 00:36:50,790 --> 00:36:55,260 maybe that's in terms of the number of ATPs degraded-- 487 00:36:55,260 --> 00:36:56,590 what do we see here? 488 00:36:56,590 --> 00:37:01,500 What is incredibly striking about these data? 489 00:37:01,500 --> 00:37:07,140 We're seeing about 600 ATPs for I27 domain degraded for this 490 00:37:07,140 --> 00:37:10,400 wild-type that's a huge number of ATPs-- 491 00:37:13,140 --> 00:37:17,010 so 600. 492 00:37:17,010 --> 00:37:20,610 What do we see for these denatured variants? 493 00:37:20,610 --> 00:37:25,680 They're all around 115 ATPs per substrate consumed here. 494 00:37:30,130 --> 00:37:33,650 So many, many ATPs are consumed here. 495 00:37:33,650 --> 00:37:37,820 Many ATPs are required to denature that native substrate. 496 00:37:37,820 --> 00:37:41,870 And it looks like many ATPs are required for translocation 497 00:37:41,870 --> 00:37:43,040 here. 498 00:37:43,040 --> 00:37:45,440 And if the substrate is less stable, what we see 499 00:37:45,440 --> 00:37:48,970 is that fewer ATPs are consumed. 500 00:37:48,970 --> 00:37:51,640 So these are all filled in within your notes. 501 00:37:51,640 --> 00:37:53,680 And there's some additional details here. 502 00:37:56,320 --> 00:38:00,550 So these data indicate that the easier the protein unfolds, 503 00:38:00,550 --> 00:38:02,740 the faster it's degraded. 504 00:38:02,740 --> 00:38:05,650 And just to reiterate, these denatured titins, 505 00:38:05,650 --> 00:38:08,230 we can think about ATP consumption 506 00:38:08,230 --> 00:38:10,900 as being indicative of that translocation event 507 00:38:10,900 --> 00:38:13,030 because they're already denatured. 508 00:38:13,030 --> 00:38:16,600 And for these native titin, the rate of ATP consumption 509 00:38:16,600 --> 00:38:19,180 is indicative of both the unfolding or denaturation 510 00:38:19,180 --> 00:38:22,980 process and the translocation process here for that. 511 00:38:26,660 --> 00:38:29,100 Here is just another way of plotting the data 512 00:38:29,100 --> 00:38:33,450 in the table, where they're just highlighting 513 00:38:33,450 --> 00:38:38,520 ATP hydrolysis and then the different types of substrates 514 00:38:38,520 --> 00:38:39,780 here. 515 00:38:39,780 --> 00:38:42,210 So we see the rate's highest for denatured 516 00:38:42,210 --> 00:38:44,130 and that it decreases with increasing 517 00:38:44,130 --> 00:38:49,890 stability of the substrate to degradation by ClpXP. 518 00:38:49,890 --> 00:38:52,740 Another interesting thing they found in these studies 519 00:38:52,740 --> 00:39:01,030 is that the ATP is consumed very linearly with time. 520 00:39:26,530 --> 00:39:37,860 So if we look at ATP consumed versus the average denaturation 521 00:39:37,860 --> 00:39:38,360 time-- 522 00:39:46,850 --> 00:39:56,150 here, wild-type, and down in this region, the mutants. 523 00:39:59,900 --> 00:40:02,360 So we have a linear relationship. 524 00:40:02,360 --> 00:40:06,980 And what came out of this is about 144 ATPs 525 00:40:06,980 --> 00:40:20,690 consumed per minute of unfolding from these experiments here. 526 00:40:27,320 --> 00:40:30,730 So what does this tell us about how ClpXP works-- 527 00:40:30,730 --> 00:40:32,450 how it works here? 528 00:40:37,400 --> 00:40:42,350 So basically, this machine has been 529 00:40:42,350 --> 00:40:46,720 described as having a relentless try and try again mechanism 530 00:40:46,720 --> 00:40:48,830 here. 531 00:40:48,830 --> 00:40:53,240 And it's effectively explained in this cartoon. 532 00:40:53,240 --> 00:40:58,200 So ClpP is omitted, but imagine it's there. 533 00:40:58,200 --> 00:41:00,950 So what's happening? 534 00:41:00,950 --> 00:41:04,340 We have some folded protein that's been condemned 535 00:41:04,340 --> 00:41:07,670 and has the ssrA tag attached. 536 00:41:07,670 --> 00:41:10,650 And so ClpXP needs to deal with it. 537 00:41:10,650 --> 00:41:13,200 There's the tag-mediated substrate binding. 538 00:41:13,200 --> 00:41:17,150 So the substrate binds, there can be ATP hydrolysis. 539 00:41:17,150 --> 00:41:23,070 And that results in ClpX trying to unfold the protein. 540 00:41:23,070 --> 00:41:27,140 But frequently, the substrate can get released. 541 00:41:27,140 --> 00:41:30,020 And this cycle of binding and pulling 542 00:41:30,020 --> 00:41:32,360 can happen many, many times. 543 00:41:32,360 --> 00:41:36,380 And that consumes a lot of ATPs here. 544 00:41:36,380 --> 00:41:38,630 And then at some point, there's going 545 00:41:38,630 --> 00:41:41,660 to be a successful unfolding event, which 546 00:41:41,660 --> 00:41:45,920 results in the polypeptide being translocated and entering 547 00:41:45,920 --> 00:41:49,340 the degradation chamber. 548 00:41:49,340 --> 00:41:52,520 So when thinking about a hard to denature substrate, 549 00:41:52,520 --> 00:41:55,640 you want to think about this substrate binding ClpX 550 00:41:55,640 --> 00:41:57,140 many times. 551 00:41:57,140 --> 00:41:59,390 There might be multiple instances of binding 552 00:41:59,390 --> 00:42:02,270 and release before it's successfully denatured 553 00:42:02,270 --> 00:42:04,220 and before translocation occurs. 554 00:42:04,220 --> 00:42:06,806 And so that uses a lot of ATPs. 555 00:42:06,806 --> 00:42:08,710 STUDENT: Does it all confine the substrate 556 00:42:08,710 --> 00:42:11,500 to many different places or just in one spot? 557 00:42:11,500 --> 00:42:16,340 PROFESSOR: So the ssr tag is what's 558 00:42:16,340 --> 00:42:18,950 going to allow it to bind to the pore. 559 00:42:18,950 --> 00:42:20,450 And recall, for instance, there can 560 00:42:20,450 --> 00:42:25,460 be the adapter protein SspB to help ssrA tag proteins make 561 00:42:25,460 --> 00:42:28,340 their way to the pore. 562 00:42:28,340 --> 00:42:33,260 So think of it less as some undesirable protein-protein 563 00:42:33,260 --> 00:42:35,750 interaction than a failed attempt 564 00:42:35,750 --> 00:42:41,120 at unfolding by this ATPase here. 565 00:42:41,120 --> 00:42:44,060 So why might ClpX want to do this? 566 00:42:52,690 --> 00:42:54,050 When we think about the cell-- 567 00:42:54,050 --> 00:42:55,120 just some possibilities? 568 00:42:55,120 --> 00:42:59,033 STUDENT: It would make sense, I guess, the more unstable 569 00:42:59,033 --> 00:43:00,950 the protein is, the easier it is to degrade it 570 00:43:00,950 --> 00:43:02,885 because proteins that are more unstable 571 00:43:02,885 --> 00:43:04,910 are already partially unfolded and are probably 572 00:43:04,910 --> 00:43:06,872 ones that need to be degraded. 573 00:43:06,872 --> 00:43:10,160 PROFESSOR: Yeah, so that's one way to think about this. 574 00:43:10,160 --> 00:43:12,110 And then maybe another way to phrase 575 00:43:12,110 --> 00:43:16,690 that is perhaps this helps to avoid jamming the protease. 576 00:43:16,690 --> 00:43:18,440 If there's things that need to be degraded 577 00:43:18,440 --> 00:43:21,290 versus other things, if you have something that's 578 00:43:21,290 --> 00:43:23,600 very difficult to degrade, you don't 579 00:43:23,600 --> 00:43:26,090 want that to block the protease such that something 580 00:43:26,090 --> 00:43:28,340 unfolded can't be dealt with. 581 00:43:28,340 --> 00:43:30,140 Also, just dealing with a mixture, 582 00:43:30,140 --> 00:43:32,690 that maybe ClpXP likes to get rid 583 00:43:32,690 --> 00:43:39,260 of the substrates that are easiest to degrade first. 584 00:43:39,260 --> 00:43:45,050 So is it energetically wasteful there-- 585 00:43:45,050 --> 00:43:46,710 just to think about. 586 00:43:46,710 --> 00:43:48,710 On one hand, it might seem like it. 587 00:43:48,710 --> 00:43:52,040 So many ATPs-- just think back to the TCA cycle, for instance, 588 00:43:52,040 --> 00:43:54,470 and how many ATPs you get from one cycle 589 00:43:54,470 --> 00:43:58,220 there versus 600 ATPs being consumed here 590 00:43:58,220 --> 00:44:01,700 for that wild-type titin domain. 591 00:44:01,700 --> 00:44:05,450 But this makes sense because in the cell, 592 00:44:05,450 --> 00:44:08,600 it does have to deal with many different types of substrates. 593 00:44:08,600 --> 00:44:11,060 And these substrates can have varying structure 594 00:44:11,060 --> 00:44:14,480 and varying stabilities. 595 00:44:14,480 --> 00:44:18,320 So how does ClpX actually work? 596 00:44:18,320 --> 00:44:23,450 What's going on with this ATP hydrolysis? 597 00:44:23,450 --> 00:44:26,750 How are denaturation and translocation coupled? 598 00:44:26,750 --> 00:44:31,160 And how do we even think about this translocation process? 599 00:44:31,160 --> 00:44:32,810 Effectively, we saw the cartoons, 600 00:44:32,810 --> 00:44:35,510 where it looked like ClpX was somehow 601 00:44:35,510 --> 00:44:38,490 pulling on this polypeptide. 602 00:44:38,490 --> 00:44:45,860 And so we'll close with some discussion about that, 603 00:44:45,860 --> 00:44:50,460 which we'll continue on Monday. 604 00:44:50,460 --> 00:45:00,560 So effectively, we have our general paradigm 605 00:45:00,560 --> 00:45:12,510 of somehow having ATP hydrolysis leading 606 00:45:12,510 --> 00:45:19,610 to conformational change that provides some mechanical work. 607 00:45:24,530 --> 00:45:36,400 And so here in this system, conformational change in ClpX 608 00:45:36,400 --> 00:45:40,750 will drive unfolding and translocation. 609 00:45:49,270 --> 00:45:51,280 And of course, the big question is how? 610 00:45:54,450 --> 00:45:59,590 And so a key observation that's not intuitive with this system 611 00:45:59,590 --> 00:46:02,020 and that we'll build upon in the first 10 minutes or so 612 00:46:02,020 --> 00:46:08,120 of Monday is the fact that ClpX is a homohexamer. 613 00:46:08,120 --> 00:46:10,870 We saw that when we went over the structure. 614 00:46:10,870 --> 00:46:14,650 But this hexamer has some inherent asymmetry to it, 615 00:46:14,650 --> 00:46:19,690 despite the fact that each subunit is the same. 616 00:46:19,690 --> 00:46:30,480 So a key observation here-- 617 00:46:30,480 --> 00:46:33,360 ClpX is homohexamer. 618 00:46:38,140 --> 00:46:43,610 But it has inherent asymmetry. 619 00:46:50,530 --> 00:46:59,760 And this asymmetry arises from nucleotide ATP binding. 620 00:47:08,070 --> 00:47:11,760 And the observation from a variety of studies 621 00:47:11,760 --> 00:47:17,750 is that ATP binds to some of the ClpX subunits but not others. 622 00:47:17,750 --> 00:47:24,900 OK And also it can bind to different subunits 623 00:47:24,900 --> 00:47:27,825 with different affinities, just as another detail. 624 00:47:32,400 --> 00:47:35,790 And what we'll see is that this is also dynamic-- 625 00:47:35,790 --> 00:47:44,470 so just some subunits. 626 00:47:48,080 --> 00:47:51,980 So although we think about this as a homohexamer in terms 627 00:47:51,980 --> 00:47:56,240 of the ATP loading at different points, 628 00:47:56,240 --> 00:48:00,200 we don't have six ATPs bound. 629 00:48:00,200 --> 00:48:02,330 And where we'll begin on Monday is 630 00:48:02,330 --> 00:48:07,190 looking at individual ClpX subunits 631 00:48:07,190 --> 00:48:12,260 and then how the ClpX subunits work together 632 00:48:12,260 --> 00:48:17,810 and lessons learned from studies there. 633 00:48:17,810 --> 00:48:19,970 So effectively, this asymmetry is 634 00:48:19,970 --> 00:48:22,190 thought to be quite important, in terms 635 00:48:22,190 --> 00:48:25,460 of how ATP hydrolysis is allowing the movements 636 00:48:25,460 --> 00:48:27,910 and activity of the ATPase.