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 00:00:27,170 --> 00:00:30,180 ELIZABETH NOLAN: OK, so we're going to get started. 9 00:00:30,180 --> 00:00:32,759 And we're going to continue on with folding. 10 00:00:32,759 --> 00:00:36,660 So we had some introduction last time 11 00:00:36,660 --> 00:00:39,450 about this module and thinking about in vitro 12 00:00:39,450 --> 00:00:40,777 versus in vivo studies. 13 00:00:40,777 --> 00:00:42,360 And where we're going to move on today 14 00:00:42,360 --> 00:00:44,520 is discussing molecular chaperones. 15 00:00:44,520 --> 00:00:48,210 And effectively, there'll be three case studies 16 00:00:48,210 --> 00:00:50,070 over the next two to two and a half 17 00:00:50,070 --> 00:00:55,670 lectures-- so trigger factor, GroEL/GroES, and DnaK/DnaJ. 18 00:00:55,670 --> 00:00:58,630 And so first is some background. 19 00:00:58,630 --> 00:01:02,220 We need to talk about what are molecular chaperones. 20 00:01:15,120 --> 00:01:19,260 And so effectively, these are proteins 21 00:01:19,260 --> 00:01:23,760 that influence protein folding within the cell. 22 00:01:23,760 --> 00:01:27,370 And they can do this by a variety of ways. 23 00:01:27,370 --> 00:01:31,440 So they can help to prevent aggregation and intermolecular 24 00:01:31,440 --> 00:01:35,910 interactions between polypeptides. 25 00:01:35,910 --> 00:01:39,840 They can facilitate folding by limiting conformational space 26 00:01:39,840 --> 00:01:42,270 and preventing side reactions. 27 00:01:42,270 --> 00:01:44,790 An important point to keep in mind throughout this 28 00:01:44,790 --> 00:01:50,330 is that these chaperone proteins bind to proteins transiently 29 00:01:50,330 --> 00:01:50,830 here. 30 00:01:53,350 --> 00:01:56,910 What are the types of processes they can assist in? 31 00:01:56,910 --> 00:01:59,530 A variety are listed here. 32 00:01:59,530 --> 00:02:01,110 And we see that it's quite broad. 33 00:02:01,110 --> 00:02:04,740 So they can help in de novo folding, so for instance, 34 00:02:04,740 --> 00:02:07,320 folding of a nascent polypeptide chain emerging 35 00:02:07,320 --> 00:02:09,660 from the ribosome. 36 00:02:09,660 --> 00:02:11,310 They can assist in refolding. 37 00:02:11,310 --> 00:02:15,570 So for instance, if proteins have unfolded or become 38 00:02:15,570 --> 00:02:19,560 aggregated because of stress, they can help here. 39 00:02:19,560 --> 00:02:24,540 They can assist in the assembly of oligomeric proteins 40 00:02:24,540 --> 00:02:26,520 in protein transport, and they also 41 00:02:26,520 --> 00:02:30,020 assist in proteolytic degradation here. 42 00:02:30,020 --> 00:02:33,120 And so we can classify these chaperones 43 00:02:33,120 --> 00:02:37,200 into three main groups depending on how they act, 44 00:02:37,200 --> 00:02:39,420 and those are listed here. 45 00:02:39,420 --> 00:02:45,270 So we can have holdases, foldases, and unfoldases. 46 00:02:45,270 --> 00:02:47,100 So something you might want to ask yourself 47 00:02:47,100 --> 00:02:49,890 as you see these different chaperone systems is to ask, 48 00:02:49,890 --> 00:02:51,360 what is the role? 49 00:02:51,360 --> 00:02:53,220 Is it one or multiple? 50 00:02:53,220 --> 00:02:57,680 So holdases help to stabilize non-native confirmations. 51 00:02:57,680 --> 00:02:59,310 So effectively, the chaperone will 52 00:02:59,310 --> 00:03:03,120 bind a polypeptide and a non-native confirmation 53 00:03:03,120 --> 00:03:06,480 and stabilize that for some period of time. 54 00:03:06,480 --> 00:03:10,200 Foldases assist in folding of a polypeptide 55 00:03:10,200 --> 00:03:12,300 to its native state. 56 00:03:12,300 --> 00:03:14,860 And unfoldases, as the name indicates, 57 00:03:14,860 --> 00:03:17,590 can help with unfolding proteins, so for instance, 58 00:03:17,590 --> 00:03:20,880 if a protein has misfolded and that needs to be undone, 59 00:03:20,880 --> 00:03:23,070 or maybe a protein needs to be extracted 60 00:03:23,070 --> 00:03:26,040 from some aggregate that's formed in in multiple proteins 61 00:03:26,040 --> 00:03:28,150 that's a problem. 62 00:03:28,150 --> 00:03:33,150 And so we're going to think about the chaperones 63 00:03:33,150 --> 00:03:37,140 in the cytoplasm in two main groups for the ones 64 00:03:37,140 --> 00:03:40,860 that interact with newly synthesized polypeptides. 65 00:03:40,860 --> 00:03:44,100 So first, we can think about trigger factor, which 66 00:03:44,100 --> 00:03:47,430 is a chaperone that's associated with the ribosome, 67 00:03:47,430 --> 00:03:48,970 as we'll see. 68 00:03:48,970 --> 00:03:52,620 So trigger factor is involved in co-translational folding, 69 00:03:52,620 --> 00:03:58,380 meaning the polypeptide is still associated with the ribosome 70 00:03:58,380 --> 00:04:00,300 and de novo folding. 71 00:04:00,300 --> 00:04:03,270 And then we'll examine some downstream cytosolic 72 00:04:03,270 --> 00:04:04,170 chaperones. 73 00:04:04,170 --> 00:04:07,710 So these are chaperones that do not bind to ribosome-- 74 00:04:07,710 --> 00:04:09,050 GroEL/GroES, DnaK/DnaJ. 75 00:04:11,870 --> 00:04:14,820 And just as a general overview of this molecular 76 00:04:14,820 --> 00:04:17,670 chaperone concept here-- 77 00:04:17,670 --> 00:04:22,050 so this is taken from the required reading-- 78 00:04:22,050 --> 00:04:25,500 effectively, what's shown in this scheme 79 00:04:25,500 --> 00:04:29,220 is a variety of different states a polypeptide 80 00:04:29,220 --> 00:04:31,020 can find itself in. 81 00:04:31,020 --> 00:04:35,040 So here we see a partially folded protein. 82 00:04:35,040 --> 00:04:39,660 This protein may form from an unfolded protein or maybe 83 00:04:39,660 --> 00:04:41,250 a native protein. 84 00:04:41,250 --> 00:04:42,630 We have an aggregate. 85 00:04:42,630 --> 00:04:45,720 And if we look down here, we're seeing the effect 86 00:04:45,720 --> 00:04:48,930 of some generalized chaperone. 87 00:04:48,930 --> 00:04:52,920 OK, so one important point to make from this 88 00:04:52,920 --> 00:04:57,520 is that the chaperone's not part of this final structure. 89 00:04:57,520 --> 00:04:59,400 It's just helping the polypeptide 90 00:04:59,400 --> 00:05:01,440 get to its native state. 91 00:05:01,440 --> 00:05:04,740 OK, and we can think about different rate constants, 92 00:05:04,740 --> 00:05:08,130 whether it be for folding or aggregation, 93 00:05:08,130 --> 00:05:12,720 chaperone binding Kon, chaperone dissociation Koff. 94 00:05:12,720 --> 00:05:14,410 So for instance, if we look here, 95 00:05:14,410 --> 00:05:17,090 we have a partially folded polypeptide. 96 00:05:17,090 --> 00:05:19,560 And imagine the chaperone binds that. 97 00:05:19,560 --> 00:05:23,200 Or maybe the chaperone binds an unfolded polypeptide. 98 00:05:23,200 --> 00:05:27,610 OK, it's going to act as a holdase or a foldase. 99 00:05:27,610 --> 00:05:29,520 And what can we see down here-- 100 00:05:29,520 --> 00:05:32,790 or an unfoldase-- what we see down here 101 00:05:32,790 --> 00:05:35,190 is an indication of an event that's 102 00:05:35,190 --> 00:05:37,980 driven by ATP hydrolysis. 103 00:05:37,980 --> 00:05:41,820 And so what we'll see and what's known 104 00:05:41,820 --> 00:05:43,950 is that many of these chaperones switch 105 00:05:43,950 --> 00:05:46,860 between low and high affinity states 106 00:05:46,860 --> 00:05:49,230 for some substrate polypeptide. 107 00:05:49,230 --> 00:05:52,200 And these low and high affinity states 108 00:05:52,200 --> 00:05:56,200 are somehow regulated by the ATP binding and hydrolysis. 109 00:05:56,200 --> 00:06:00,400 So here, for instance, we see that step. 110 00:06:00,400 --> 00:06:03,630 And imagine a Koff getting us back into this direction 111 00:06:03,630 --> 00:06:05,160 here, right? 112 00:06:05,160 --> 00:06:08,040 So you can begin to ask yourself questions like, 113 00:06:08,040 --> 00:06:11,340 under what conditions and terms of these rate constants 114 00:06:11,340 --> 00:06:13,500 is folding efficient? 115 00:06:13,500 --> 00:06:16,830 When would a chaperone act as a holdase? 116 00:06:16,830 --> 00:06:18,960 When would aggregation occur? 117 00:06:18,960 --> 00:06:22,650 So aggregation would occur if this Kagg 118 00:06:22,650 --> 00:06:27,780 is much greater than, say, for instance, Kon here 119 00:06:27,780 --> 00:06:31,860 to work systematically through this scheme. 120 00:06:31,860 --> 00:06:34,320 And here are just some points and words 121 00:06:34,320 --> 00:06:37,200 related to that scheme and things 122 00:06:37,200 --> 00:06:40,890 to think about from a broader picture. 123 00:06:40,890 --> 00:06:43,560 So in terms of the systems we're going 124 00:06:43,560 --> 00:06:49,500 to examine in the cytoplasm, this is the overview slide. 125 00:06:49,500 --> 00:06:53,100 And where we're going to begin in this overview 126 00:06:53,100 --> 00:06:55,290 is with the ribosome. 127 00:06:55,290 --> 00:06:58,740 And we see that in red here we have a nascent polypeptide 128 00:06:58,740 --> 00:07:00,560 chain emerging. 129 00:07:00,560 --> 00:07:06,180 OK, so what does this scheme indicate? 130 00:07:06,180 --> 00:07:10,020 What we see is that here is the player trigger 131 00:07:10,020 --> 00:07:14,670 factor, which is involved in co-translational folding. 132 00:07:14,670 --> 00:07:19,380 And we see that about 70% of nascent polypeptides 133 00:07:19,380 --> 00:07:21,240 interact with trigger factor. 134 00:07:21,240 --> 00:07:24,810 And these can arrive in a native conformation. 135 00:07:24,810 --> 00:07:28,110 We see there's two other systems here. 136 00:07:28,110 --> 00:07:31,860 So on the right, we have GroES and GroEL. 137 00:07:31,860 --> 00:07:37,680 So GroEL provides post-translational folding. 138 00:07:37,680 --> 00:07:41,550 We look and see about 10% to 15% of peptides in the cell 139 00:07:41,550 --> 00:07:44,340 interact with GroEL/GroES. 140 00:07:44,340 --> 00:07:48,160 And as we'll see, it provides this folding chamber 141 00:07:48,160 --> 00:07:51,760 on a protected space. 142 00:07:51,760 --> 00:07:54,940 We also see here that this system uses ATP. 143 00:07:54,940 --> 00:07:59,340 OK, and here we have another two players, DnaK 144 00:07:59,340 --> 00:08:01,860 and its co-chaperone DnaJ. 145 00:08:01,860 --> 00:08:05,550 And we see they're binding to some sort of polypeptide 146 00:08:05,550 --> 00:08:08,820 in a manner that's different than GroEL/GroES. 147 00:08:08,820 --> 00:08:11,880 OK, about 5% to 18% of polypeptides 148 00:08:11,880 --> 00:08:14,490 come into contact with these two players. 149 00:08:14,490 --> 00:08:17,820 We also see this system as ATP-dependent, 150 00:08:17,820 --> 00:08:19,710 and there's another player, GrpE, 151 00:08:19,710 --> 00:08:21,720 which we'll see is the nucleotide exchange 152 00:08:21,720 --> 00:08:24,080 factor needed here. 153 00:08:24,080 --> 00:08:27,780 OK, so we see that maybe there's some crosstalk here. 154 00:08:27,780 --> 00:08:33,480 And here we have some needed native polypeptides. 155 00:08:33,480 --> 00:08:35,730 So just some things to keep in mind-- 156 00:08:35,730 --> 00:08:38,700 it's important to think about concentrations 157 00:08:38,700 --> 00:08:42,640 and some approximate concentrations are listed here. 158 00:08:42,640 --> 00:08:44,490 If we're thinking about the ribosome 159 00:08:44,490 --> 00:08:50,010 DnaK/DnaJ, GroEL, and trigger factor. 160 00:08:50,010 --> 00:08:54,060 Just to note that many chaperones are also 161 00:08:54,060 --> 00:08:56,100 called Heat Shock Proteins, and this 162 00:08:56,100 --> 00:08:59,040 is because their expression increases with increased 163 00:08:59,040 --> 00:09:00,810 temperature or stress. 164 00:09:00,810 --> 00:09:06,720 So Hsp70, Hsp60, that's for heat shock protein. 165 00:09:06,720 --> 00:09:09,630 So where we're going to begin is with an overview 166 00:09:09,630 --> 00:09:10,900 of trigger factor. 167 00:09:10,900 --> 00:09:11,515 Yes? 168 00:09:11,515 --> 00:09:12,598 AUDIENCE: I wanted to ask. 169 00:09:12,598 --> 00:09:18,550 What do you mean by native and this protein 170 00:09:18,550 --> 00:09:20,540 exist in several conformations in this slide? 171 00:09:20,540 --> 00:09:22,540 ELIZABETH NOLAN: So proteins are dynamic, right? 172 00:09:22,540 --> 00:09:23,640 We know that. 173 00:09:23,640 --> 00:09:27,600 So native means a native fold, so a native state 174 00:09:27,600 --> 00:09:31,410 of this protein as opposed to the protein being unfolded 175 00:09:31,410 --> 00:09:34,470 if it's supposed to be globular or being 176 00:09:34,470 --> 00:09:38,190 some undesirable oligomer aggregate. 177 00:09:38,190 --> 00:09:42,000 So when this polypeptide comes off the ribosome, 178 00:09:42,000 --> 00:09:44,280 that's a linear sequence of amino acids. 179 00:09:44,280 --> 00:09:47,250 And it needs to adopt its appropriate conformation 180 00:09:47,250 --> 00:09:49,200 to do its job, OK? 181 00:09:49,200 --> 00:09:50,760 And as I said last time, we're not 182 00:09:50,760 --> 00:09:52,800 discussing natively unfolded proteins 183 00:09:52,800 --> 00:09:54,300 in the context of this class. 184 00:09:54,300 --> 00:09:58,196 AUDIENCE: So proteins can have many different native receptors 185 00:09:58,196 --> 00:09:59,170 in this slide? 186 00:09:59,170 --> 00:10:00,780 ELIZABETH NOLAN: Yes, they're dynamic. 187 00:10:00,780 --> 00:10:03,420 But there is going to be, like if you need a beta sheet, 188 00:10:03,420 --> 00:10:06,270 a domain that has beta sheets, for instance, that 189 00:10:06,270 --> 00:10:08,640 needs to fold and form there. 190 00:10:08,640 --> 00:10:12,210 So we can discuss further if you have more questions about that. 191 00:10:12,210 --> 00:10:13,980 But think about ubiquitin, for instance, 192 00:10:13,980 --> 00:10:15,180 from recitation week one. 193 00:10:15,180 --> 00:10:19,200 That had a very defined shape, right? 194 00:10:19,200 --> 00:10:25,336 So it's native fold from looking at that PDB file. 195 00:10:25,336 --> 00:10:27,585 AUDIENCE: OK, I think I just need to understand 196 00:10:27,585 --> 00:10:28,543 in that previous slide. 197 00:10:28,543 --> 00:10:31,860 When I talk about one protein here, right? 198 00:10:31,860 --> 00:10:33,805 70% percent of the proteins-- 199 00:10:33,805 --> 00:10:35,430 ELIZABETH NOLAN: Yeah, this is thinking 200 00:10:35,430 --> 00:10:37,795 about all the proteins and all the peptides in the cell. 201 00:10:37,795 --> 00:10:39,920 AUDIENCE: OK, I thought it was one type of protein. 202 00:10:39,920 --> 00:10:41,420 ELIZABETH NOLAN: No, this is looking 203 00:10:41,420 --> 00:10:42,810 at proteins in broad terms. 204 00:10:42,810 --> 00:10:44,790 And so what we'll see as we move forward 205 00:10:44,790 --> 00:10:47,430 is that certain types of proteins 206 00:10:47,430 --> 00:10:49,980 interact with GroEL where others don't, right? 207 00:10:49,980 --> 00:10:52,530 Trigger factor interacts with many, many of them 208 00:10:52,530 --> 00:10:54,300 because it's associated with the ribosome, 209 00:10:54,300 --> 00:10:59,160 and the ribosome's synthesizing all polypeptide chains there. 210 00:10:59,160 --> 00:11:02,220 OK, so we're going to start with trigger factor. 211 00:11:02,220 --> 00:11:05,970 And the first thing just to be aware of when thinking about 212 00:11:05,970 --> 00:11:07,770 trigger factor and where it acts-- 213 00:11:07,770 --> 00:11:11,250 so we saw it sitting on top of the exit tunnel of the 50S 214 00:11:11,250 --> 00:11:13,340 in the prior slide-- 215 00:11:13,340 --> 00:11:16,430 is that there is a lot of things happening near the exit 216 00:11:16,430 --> 00:11:18,320 tunnel of the ribosome. 217 00:11:18,320 --> 00:11:20,930 OK, so here we have our 70S ribosome. 218 00:11:20,930 --> 00:11:22,850 Here's the polypeptide coming out. 219 00:11:22,850 --> 00:11:25,420 A few proteins are indicated. 220 00:11:25,420 --> 00:11:27,200 In addition to trigger factor, just 221 00:11:27,200 --> 00:11:30,080 be aware that there's other players here. 222 00:11:30,080 --> 00:11:32,960 OK, one of these is Signal Recognition Protocol, which 223 00:11:32,960 --> 00:11:35,720 I mentioned briefly last time. 224 00:11:35,720 --> 00:11:39,980 This is involved in delivering membrane proteins 225 00:11:39,980 --> 00:11:41,490 to their destination. 226 00:11:41,490 --> 00:11:44,240 We also have enzymes that do work here, 227 00:11:44,240 --> 00:11:46,070 whether it's an enzyme for removing 228 00:11:46,070 --> 00:11:49,370 the N-terminal methionine, enzymes for deformulation 229 00:11:49,370 --> 00:11:52,820 of that N-terminal methionine, et cetera. 230 00:11:52,820 --> 00:11:55,610 So somehow, trigger factor needs to work 231 00:11:55,610 --> 00:11:59,990 in the presence of these other constituents there. 232 00:12:09,820 --> 00:12:13,840 OK, so when we think about trigger factor, what 233 00:12:13,840 --> 00:12:16,010 you want to think about is a protein 234 00:12:16,010 --> 00:12:19,260 of about 50-kDa that's shaped like a dragon. 235 00:12:25,370 --> 00:12:28,380 OK, this is ATP-independent. 236 00:12:31,650 --> 00:12:36,420 So what I said earlier about low and high affinity states 237 00:12:36,420 --> 00:12:38,550 and switching between these states being driven 238 00:12:38,550 --> 00:12:40,710 by ATP binding and hydrolysis, that 239 00:12:40,710 --> 00:12:42,330 does not apply for trigger factor. 240 00:12:42,330 --> 00:12:46,060 It's the exception in what will be presented in this course. 241 00:12:46,060 --> 00:12:47,940 And it's associated with the ribosome. 242 00:12:53,100 --> 00:13:04,230 And what trigger factor does is it provides a folding cavity 243 00:13:04,230 --> 00:13:09,670 or cradle over the exit tunnel. 244 00:13:15,200 --> 00:13:18,170 And by doing so, it gives this emerging polypeptide 245 00:13:18,170 --> 00:13:21,440 a protected space to begin to fold, 246 00:13:21,440 --> 00:13:25,230 so reduction of intermolecular interactions. 247 00:13:25,230 --> 00:13:28,100 So if we take a look at trigger factor, 248 00:13:28,100 --> 00:13:30,090 one depiction is shown here. 249 00:13:30,090 --> 00:13:32,390 And as I said, think about a dragon. 250 00:13:32,390 --> 00:13:36,860 And it's actually described as having a head, arms, 251 00:13:36,860 --> 00:13:38,990 and a tail. 252 00:13:38,990 --> 00:13:40,970 And so we can think about this also 253 00:13:40,970 --> 00:13:43,850 in terms of N and C-terminus. 254 00:13:43,850 --> 00:13:45,710 The region of trigger factor that 255 00:13:45,710 --> 00:13:48,950 interacts with the ribosome and binds the ribosome 256 00:13:48,950 --> 00:13:52,560 is down here in the tail region. 257 00:13:52,560 --> 00:13:55,010 And so what trigger factor does and what's 258 00:13:55,010 --> 00:13:57,770 indicated by the cartoon you've already seen 259 00:13:57,770 --> 00:14:01,220 is that it associates with the translating ribosome 260 00:14:01,220 --> 00:14:03,380 with a one-to-one stoichiometry. 261 00:14:03,380 --> 00:14:06,440 So think about having one trigger factor over that exit 262 00:14:06,440 --> 00:14:07,990 tunnel here. 263 00:14:07,990 --> 00:14:10,400 And as you can see, these different domains 264 00:14:10,400 --> 00:14:13,340 also have some additional activities. 265 00:14:13,340 --> 00:14:15,560 And the main chaperone activity is 266 00:14:15,560 --> 00:14:19,331 attributed to the C-terminal region here I've color-coded. 267 00:14:22,160 --> 00:14:26,580 So what are some characteristics of trigger factor? 268 00:14:26,580 --> 00:14:29,480 If we look at the surface and consider 269 00:14:29,480 --> 00:14:32,870 where different types of amino acids are found, 270 00:14:32,870 --> 00:14:36,440 so whether they have polar or non-polar residues, 271 00:14:36,440 --> 00:14:38,780 that's depicted here. 272 00:14:38,780 --> 00:14:41,248 OK, what do we see in this depiction? 273 00:14:41,248 --> 00:14:42,290 Where are these residues? 274 00:14:45,110 --> 00:14:50,260 Is a given type of residue clustered in any one spot? 275 00:14:50,260 --> 00:14:52,110 Yeah, I see some heads shaking no. 276 00:14:52,110 --> 00:14:53,070 No, right. 277 00:14:53,070 --> 00:14:55,230 They're distributed all about. 278 00:14:55,230 --> 00:14:57,840 We see non-polar and polar residues 279 00:14:57,840 --> 00:15:03,690 distributed across the surface of trigger factor here. 280 00:15:03,690 --> 00:15:07,020 And so why might this be? 281 00:15:07,020 --> 00:15:10,620 What's thought is that, effectively, trigger factor 282 00:15:10,620 --> 00:15:12,810 uses its entire inner cavity-- 283 00:15:12,810 --> 00:15:15,270 and you'll see how that forms in a minute-- 284 00:15:15,270 --> 00:15:16,740 for substrate accommodation. 285 00:15:16,740 --> 00:15:19,650 And you can imagine that all of these different polypeptides 286 00:15:19,650 --> 00:15:21,960 emerging from the ribosome have different amino acid 287 00:15:21,960 --> 00:15:23,400 compositions, right? 288 00:15:23,400 --> 00:15:26,070 So this allows it to be relatively 289 00:15:26,070 --> 00:15:29,160 versatile from that perspective. 290 00:15:29,160 --> 00:15:32,130 If we take another look at structure 291 00:15:32,130 --> 00:15:34,590 and think about how trigger factor binds 292 00:15:34,590 --> 00:15:38,880 to the ribosome, what's found here, 293 00:15:38,880 --> 00:15:40,680 so we're looking at structures of trigger 294 00:15:40,680 --> 00:15:43,500 factor bound in orange and unbound 295 00:15:43,500 --> 00:15:44,820 in green to the ribosome. 296 00:15:44,820 --> 00:15:47,550 And the ribosome's omitted for clarity. 297 00:15:47,550 --> 00:15:50,190 There's a helix-loop-helix region 298 00:15:50,190 --> 00:15:53,430 that is involved in that interaction. 299 00:15:53,430 --> 00:15:57,270 And what's found is that when trigger factor is bound, 300 00:15:57,270 --> 00:15:58,380 it's quite dynamic. 301 00:15:58,380 --> 00:16:02,850 And it can swivel around this ribosome binding site 302 00:16:02,850 --> 00:16:05,700 by about 10 degrees in every direction. 303 00:16:05,700 --> 00:16:10,680 So why might that be important? 304 00:16:10,680 --> 00:16:13,590 One, this flexibility may allow it 305 00:16:13,590 --> 00:16:17,460 to accommodate many different polypeptides that 306 00:16:17,460 --> 00:16:19,320 are emerging from the ribosome. 307 00:16:19,320 --> 00:16:22,110 And it may also facilitate its coexistence 308 00:16:22,110 --> 00:16:24,660 with those other proteins and enzymes 309 00:16:24,660 --> 00:16:28,020 that are acting by the exit tunnel 310 00:16:28,020 --> 00:16:30,570 that we saw on the prior slide. 311 00:16:30,570 --> 00:16:35,460 OK, so here is just a model attempting 312 00:16:35,460 --> 00:16:38,190 to show the different ways and degrees 313 00:16:38,190 --> 00:16:42,750 to which trigger factor can move from various structural studies 314 00:16:42,750 --> 00:16:44,850 when attached to this ribosome here. 315 00:16:48,640 --> 00:16:55,330 So this N-terminal domain, I have protein L23 listed here. 316 00:16:55,330 --> 00:17:00,220 It also contacts the 23S ribosomal RNA and the protein 317 00:17:00,220 --> 00:17:01,840 L29. 318 00:17:01,840 --> 00:17:05,740 And what's found is that some salt bridge interaction 319 00:17:05,740 --> 00:17:06,760 is important. 320 00:17:06,760 --> 00:17:10,380 And we'll see that in a moment. 321 00:17:10,380 --> 00:17:14,890 Here, if we look at a depiction of trigger factor actually 322 00:17:14,890 --> 00:17:18,760 bound to the 50S, so here we have the 50S. 323 00:17:18,760 --> 00:17:20,470 Here's the exit tunnel. 324 00:17:20,470 --> 00:17:24,460 And here's our dragon-shaped molecule sitting. 325 00:17:24,460 --> 00:17:25,359 I like to say on top. 326 00:17:25,359 --> 00:17:26,680 I guess it's on bottom here. 327 00:17:26,680 --> 00:17:29,440 But anyhow, the polypeptide's coming out, 328 00:17:29,440 --> 00:17:31,990 and it has this cavity where it's 329 00:17:31,990 --> 00:17:36,490 protected from all of the other constituents in the cell. 330 00:17:36,490 --> 00:17:39,310 And here's just a rotated view showing it 331 00:17:39,310 --> 00:17:44,140 on top-- so tail region, head, and arms. 332 00:17:44,140 --> 00:17:47,020 So we have this cradle over the exit tunnel. 333 00:17:47,020 --> 00:17:49,450 It's giving a protecting space for folding 334 00:17:49,450 --> 00:17:53,020 of that nascent polypeptide chain. 335 00:17:53,020 --> 00:17:59,190 If we just look at a little more detail here, what do we see? 336 00:17:59,190 --> 00:18:02,660 So there's a salt bridge between an arginine residue, 337 00:18:02,660 --> 00:18:06,520 arginine 45 of trigger factor, and glutamate, 338 00:18:06,520 --> 00:18:11,980 glutamate 13 of the ribosomal L23 that forms a salt bridge. 339 00:18:11,980 --> 00:18:15,940 So in this depiction, we have trigger factor in red. 340 00:18:15,940 --> 00:18:18,710 We have L23 in green. 341 00:18:18,710 --> 00:18:20,380 OK, and so you need to be thinking 342 00:18:20,380 --> 00:18:24,070 about the amino acid side chains here, and that's something, 343 00:18:24,070 --> 00:18:26,578 too, Joanne and I want to stress a bit after recitation 344 00:18:26,578 --> 00:18:28,120 last week is, really, in this course, 345 00:18:28,120 --> 00:18:31,960 the importance of thinking back to the chemical structures 346 00:18:31,960 --> 00:18:35,320 and properties of the molecules that 347 00:18:35,320 --> 00:18:37,450 come up within this course-- 348 00:18:37,450 --> 00:18:40,420 so positive charge, negative charge, that interaction 349 00:18:40,420 --> 00:18:42,310 here for that. 350 00:18:47,530 --> 00:18:51,710 So what happens when a polypeptide is 351 00:18:51,710 --> 00:18:54,050 emerging from the exit tunnel and it 352 00:18:54,050 --> 00:18:56,750 encounters trigger factor? 353 00:18:56,750 --> 00:18:59,330 There's many possibilities. 354 00:18:59,330 --> 00:19:03,290 And as I said, trigger factor is dynamic. 355 00:19:03,290 --> 00:19:06,440 And an interesting point about this protein 356 00:19:06,440 --> 00:19:18,420 is that trigger factor can differentiate 357 00:19:18,420 --> 00:19:31,710 between vacant and translating ribosomes, 358 00:19:31,710 --> 00:19:36,070 OK, and so what's found from in vitro studies 359 00:19:36,070 --> 00:19:37,990 is that the binding affinity, which 360 00:19:37,990 --> 00:19:40,270 I'll describe as a dissociation constant which 361 00:19:40,270 --> 00:19:44,140 is 1 over the Ka of trigger factor for the ribosome 362 00:19:44,140 --> 00:19:46,600 varies by several orders of magnitude 363 00:19:46,600 --> 00:19:50,240 depending on whether or not the ribosome's translating. 364 00:19:50,240 --> 00:19:56,560 So we have the Kd measured on the order of 1 to 2 micromolar 365 00:19:56,560 --> 00:20:10,180 if the ribosome is vacant, and a Kd of about 40 to 70 nanomolar 366 00:20:10,180 --> 00:20:20,000 for a translating ribosome. 367 00:20:23,740 --> 00:20:26,920 OK, and just in recitation 10, we'll 368 00:20:26,920 --> 00:20:29,350 talk about binding studies more. 369 00:20:29,350 --> 00:20:33,010 But just if needed for review, if we're 370 00:20:33,010 --> 00:20:39,910 thinking about A plus B going to AB, we have Kon and Koff. 371 00:20:39,910 --> 00:20:52,830 And Kd is Koff over Kon, and Kd is 1 over the Ka here. 372 00:20:52,830 --> 00:21:00,160 So let's look at some aspects of a model for a trigger factor 373 00:21:00,160 --> 00:21:02,560 dynamics during translation. 374 00:21:02,560 --> 00:21:04,240 So as I said, it can differentiate 375 00:21:04,240 --> 00:21:07,690 the vacant and translating ribosomes. 376 00:21:07,690 --> 00:21:10,300 What's been found from in vitro studies 377 00:21:10,300 --> 00:21:13,870 is that the mean residence time on the ribosome 378 00:21:13,870 --> 00:21:16,550 is about 10 seconds. 379 00:21:16,550 --> 00:21:20,180 So what are the possibilities? 380 00:21:20,180 --> 00:21:24,160 One, trigger factor can bind to a vacant ribosome, 381 00:21:24,160 --> 00:21:26,000 and that's shown here. 382 00:21:26,000 --> 00:21:28,390 And it can bind to a translating ribosome, 383 00:21:28,390 --> 00:21:33,700 and it does this with higher affinity, so greater Kon. 384 00:21:33,700 --> 00:21:36,070 So what happens after trigger factor binds 385 00:21:36,070 --> 00:21:37,840 to a translating ribosome? 386 00:21:37,840 --> 00:21:40,660 What we see is that the nascent polypeptide 387 00:21:40,660 --> 00:21:41,960 chain is coming out. 388 00:21:41,960 --> 00:21:43,870 And in this cartoon, what's depicted 389 00:21:43,870 --> 00:21:49,150 is that it's beginning to fold in this protected region here 390 00:21:49,150 --> 00:21:51,850 made by the trigger factor cradle. 391 00:21:51,850 --> 00:21:54,070 And what we see from this point is that there's 392 00:21:54,070 --> 00:21:56,300 three possibilities. 393 00:21:56,300 --> 00:21:59,860 So if we look first on the left, what happens? 394 00:21:59,860 --> 00:22:03,280 Trigger factor dissociated from that polypeptide that's 395 00:22:03,280 --> 00:22:05,860 emerging from the ribosome. 396 00:22:05,860 --> 00:22:10,240 So recall these chaperones bind and release the polypeptides. 397 00:22:10,240 --> 00:22:12,640 In this case, it's left. 398 00:22:12,640 --> 00:22:14,530 There's some folding that's happened, 399 00:22:14,530 --> 00:22:18,130 and this peptide is still associated with the ribosome. 400 00:22:18,130 --> 00:22:19,900 So what might happen next? 401 00:22:19,900 --> 00:22:22,450 Maybe this polypeptide has the ability 402 00:22:22,450 --> 00:22:25,480 to reach its native state without the help of trigger 403 00:22:25,480 --> 00:22:27,010 factor anymore. 404 00:22:27,010 --> 00:22:28,270 So that's shown here. 405 00:22:28,270 --> 00:22:30,460 It's released, and it's folded. 406 00:22:30,460 --> 00:22:33,640 Maybe some other chaperones in the cytoplasm helped with that, 407 00:22:33,640 --> 00:22:35,750 but it's not shown here. 408 00:22:35,750 --> 00:22:38,800 Alternatively, maybe trigger factor binds again. 409 00:22:38,800 --> 00:22:41,740 So maybe this is one domain, and then somewhere else, 410 00:22:41,740 --> 00:22:44,500 there's some other region that needs some help with folding. 411 00:22:44,500 --> 00:22:45,950 And we see that here. 412 00:22:45,950 --> 00:22:48,460 So it can bind and release the same polypeptide 413 00:22:48,460 --> 00:22:51,040 more than once. 414 00:22:51,040 --> 00:22:53,050 What are our other options? 415 00:22:53,050 --> 00:22:55,630 So maybe trigger factor, after being here, 416 00:22:55,630 --> 00:22:58,050 remains bound to the ribosome, and the polypeptide 417 00:22:58,050 --> 00:23:00,570 is released. 418 00:23:00,570 --> 00:23:02,020 Or look what happens here. 419 00:23:02,020 --> 00:23:04,840 We have trigger factor bound. 420 00:23:04,840 --> 00:23:07,000 We see release of the polypeptide 421 00:23:07,000 --> 00:23:09,130 with trigger factor bound, or here we 422 00:23:09,130 --> 00:23:10,720 see that there's even two trigger 423 00:23:10,720 --> 00:23:13,330 factors bound to the same polypeptide emerging 424 00:23:13,330 --> 00:23:14,500 from the ribosome. 425 00:23:17,580 --> 00:23:20,610 And just thinking about this from the perspective 426 00:23:20,610 --> 00:23:22,650 of the number of different polypeptides 427 00:23:22,650 --> 00:23:25,410 that are synthesized by an organism, 428 00:23:25,410 --> 00:23:29,160 all different lengths, all different levels of complexity, 429 00:23:29,160 --> 00:23:33,930 it's not too surprising that there's various possibilities 430 00:23:33,930 --> 00:23:34,450 here. 431 00:23:34,450 --> 00:23:36,150 So again, if you're presented with data, 432 00:23:36,150 --> 00:23:38,910 you need to ask, what does the data say? 433 00:23:38,910 --> 00:23:43,410 And what type of particular aspect of this model 434 00:23:43,410 --> 00:23:44,310 does it support? 435 00:23:44,310 --> 00:23:45,472 Yeah? 436 00:23:45,472 --> 00:23:48,960 AUDIENCE: How often is the ribosome actually vacant? 437 00:23:48,960 --> 00:23:51,620 ELIZABETH NOLAN: How often is the ribosome vacant? 438 00:23:51,620 --> 00:23:54,140 Yeah, I don't know how often the ribosome is vacant. 439 00:23:54,140 --> 00:24:00,030 So in vivo, in your test tube, you 440 00:24:00,030 --> 00:24:01,590 can completely control that, which 441 00:24:01,590 --> 00:24:04,410 is what's going to give some of these data here. 442 00:24:04,410 --> 00:24:05,910 Joanne, do you know? 443 00:24:05,910 --> 00:24:07,120 No. 444 00:24:07,120 --> 00:24:11,070 Yeah, anybody know? 445 00:24:11,070 --> 00:24:12,170 I don't know, right? 446 00:24:12,170 --> 00:24:16,596 So does it make sense to have many vacant ribosomes? 447 00:24:16,596 --> 00:24:18,660 AUDIENCE: Are there more vacant ribosomes maybe 448 00:24:18,660 --> 00:24:20,672 like floating around than there are 449 00:24:20,672 --> 00:24:24,450 membranes bound [INAUDIBLE]? 450 00:24:24,450 --> 00:24:28,140 ELIZABETH NOLAN: So I think that's a can of worms we're not 451 00:24:28,140 --> 00:24:30,090 going to go down right now in terms 452 00:24:30,090 --> 00:24:33,870 of where the ribosome is here. 453 00:24:33,870 --> 00:24:37,710 So let's look at a functional cycle. 454 00:24:37,710 --> 00:24:42,270 This is just another depiction of a potential functional cycle 455 00:24:42,270 --> 00:24:47,070 where we have the ribosome bound to mRNA. 456 00:24:47,070 --> 00:24:48,930 There's a nascent chain. 457 00:24:48,930 --> 00:24:53,460 Here we see several trigger factors bound, 458 00:24:53,460 --> 00:24:54,600 and we see options. 459 00:24:54,600 --> 00:24:57,510 So either the native fold, or maybe there 460 00:24:57,510 --> 00:25:00,750 needs to be some work of downstream chaperones, right? 461 00:25:00,750 --> 00:25:04,500 And at some point, triggered factor will be dissociated, 462 00:25:04,500 --> 00:25:07,080 and it can come around and rebind again. 463 00:25:07,080 --> 00:25:09,870 So there is some evidence the formation of a trigger factor 464 00:25:09,870 --> 00:25:13,470 dimer when it is not with the ribosome. 465 00:25:13,470 --> 00:25:16,820 We don't need to worry about that detail too much 466 00:25:16,820 --> 00:25:18,960 for our thinking about what's happening here, 467 00:25:18,960 --> 00:25:23,610 because this is a one-to-one stoichiometry. 468 00:25:23,610 --> 00:25:26,647 So how is trigger factor influencing 469 00:25:26,647 --> 00:25:27,480 the folding process? 470 00:25:32,340 --> 00:25:40,460 If we think about foldase, unfoldase, and holdase, 471 00:25:40,460 --> 00:25:43,880 so these cartoons show that some folding is happening 472 00:25:43,880 --> 00:25:48,140 in that cradle, especially the ones we saw before, right? 473 00:25:48,140 --> 00:25:52,100 And that's perfectly reasonable that somehow trigger factor 474 00:25:52,100 --> 00:25:56,240 is allowing or accelerating productive co-translational 475 00:25:56,240 --> 00:25:57,890 folding of that polypeptide. 476 00:25:57,890 --> 00:26:02,090 So from that perspective, it would be a foldase. 477 00:26:02,090 --> 00:26:05,570 Is it possible that it's also a holdase? 478 00:26:05,570 --> 00:26:08,570 And could trigger factor, in certain cases, 479 00:26:08,570 --> 00:26:12,080 keep nascent chains unfolded? 480 00:26:12,080 --> 00:26:16,490 Maybe to help prevent premature folding that 481 00:26:16,490 --> 00:26:20,510 would be an error during polypeptide synthesis, that's 482 00:26:20,510 --> 00:26:22,130 another possibility. 483 00:26:22,130 --> 00:26:24,440 And they're not mutually exclusive, right? 484 00:26:24,440 --> 00:26:27,410 So again, it's a question of an individual system 485 00:26:27,410 --> 00:26:29,760 and looking at the data. 486 00:26:29,760 --> 00:26:32,540 So the behavior may depend on the circumstance 487 00:26:32,540 --> 00:26:34,470 in the polypeptide chain. 488 00:26:34,470 --> 00:26:34,970 Rebecca? 489 00:26:34,970 --> 00:26:36,230 AUDIENCE: I'm just curious. 490 00:26:36,230 --> 00:26:38,100 So when we're talking about it acting 491 00:26:38,100 --> 00:26:45,680 as a foldase, mechanistically, is the trigger factor 492 00:26:45,680 --> 00:26:47,650 physically interacting with and promoting 493 00:26:47,650 --> 00:26:48,620 a certain conformation? 494 00:26:48,620 --> 00:26:50,400 Or is it just providing a space where 495 00:26:50,400 --> 00:26:51,567 everything else is isolated? 496 00:26:51,567 --> 00:26:52,730 Or do we even know? 497 00:26:52,730 --> 00:26:55,520 ELIZABETH NOLAN: Yeah, so do we even know? 498 00:26:55,520 --> 00:26:58,100 So this is something we'll talk more about in the context 499 00:26:58,100 --> 00:27:00,080 of the chamber GroEL. 500 00:27:00,080 --> 00:27:02,360 But what are the possibilities? 501 00:27:02,360 --> 00:27:04,940 One is that trigger factor is just 502 00:27:04,940 --> 00:27:08,780 providing a safe place for this polypeptide 503 00:27:08,780 --> 00:27:11,090 to fold to its native conformation. 504 00:27:11,090 --> 00:27:14,090 Because recall last time, we discussed the primary sequence 505 00:27:14,090 --> 00:27:17,120 and how primary sequence can dictate the fold 506 00:27:17,120 --> 00:27:19,490 and what's thermodynamically most stable, right? 507 00:27:19,490 --> 00:27:22,430 But in the cell, the cell is very crowded, right? 508 00:27:22,430 --> 00:27:25,370 So trigger factor can protect this polypeptide 509 00:27:25,370 --> 00:27:27,620 from all those other constituents in the cell that 510 00:27:27,620 --> 00:27:31,220 might cause unwanted intermolecular 511 00:27:31,220 --> 00:27:33,140 interactions, for instance, and cause 512 00:27:33,140 --> 00:27:35,810 a different folding trajectory. 513 00:27:35,810 --> 00:27:38,720 Is it possible that the cavity wall of trigger factor 514 00:27:38,720 --> 00:27:41,870 could influence that energy landscape? 515 00:27:41,870 --> 00:27:47,180 So that's the other aspect of your question. 516 00:27:47,180 --> 00:27:50,450 Is it an Anfinsen cage, so just allowing folding? 517 00:27:50,450 --> 00:27:53,030 Or is it actually affecting the landscape? 518 00:27:53,030 --> 00:27:55,190 I don't know if we're suggesting that it 519 00:27:55,190 --> 00:27:57,710 influences the landscape, the energy landscape. 520 00:27:57,710 --> 00:28:00,420 But that doesn't mean that literature is not 521 00:28:00,420 --> 00:28:03,200 out there for that. 522 00:28:03,200 --> 00:28:07,790 So I think of it typically as a cradle. 523 00:28:07,790 --> 00:28:11,600 And as I said, we'll come back to this idea with GroEL 524 00:28:11,600 --> 00:28:14,540 where there have been studies and people arguing one 525 00:28:14,540 --> 00:28:15,230 over the other. 526 00:28:17,870 --> 00:28:23,570 OK, so with that, we're going to leave trigger factor 527 00:28:23,570 --> 00:28:30,380 and move to the macromolecular machine, GroEL/GroES. 528 00:28:30,380 --> 00:28:47,740 And so GroEL falls into a subset of chaperones 529 00:28:47,740 --> 00:28:53,980 that are called chaperonins. 530 00:28:53,980 --> 00:28:58,210 OK, and these are chaperones that 531 00:28:58,210 --> 00:29:02,250 are essential for viability in all tested cases. 532 00:29:02,250 --> 00:29:04,360 OK, so that tells you this machinery 533 00:29:04,360 --> 00:29:07,120 is really important for the cell and must 534 00:29:07,120 --> 00:29:13,270 be involved in folding of some important players here. 535 00:29:13,270 --> 00:29:17,500 So in terms of GroEL/GroES, what do we have? 536 00:29:17,500 --> 00:29:20,950 I can describe this as bullet-shaped. 537 00:29:28,720 --> 00:29:30,440 a bullet-shaped folding machine. 538 00:29:33,210 --> 00:29:38,870 And so GroEL is the chaperone, and GroES is the co-chaperone. 539 00:29:38,870 --> 00:29:40,710 And they work together. 540 00:29:40,710 --> 00:29:53,040 And so what we have if we draw this in cartoon form is 541 00:29:53,040 --> 00:30:00,320 we have GroES, and we can describe GroES 542 00:30:00,320 --> 00:30:05,360 as the lid of the folding chamber. 543 00:30:05,360 --> 00:30:07,780 And here we have GroEL. 544 00:30:11,950 --> 00:30:16,505 OK, and what GroEL is, this gives us cavities for folding. 545 00:30:24,100 --> 00:30:29,610 OK, and we can think of it like a barrel. 546 00:30:29,610 --> 00:30:33,450 And as drawn, we see two pieces here. 547 00:30:33,450 --> 00:30:35,430 And as we'll look further, we'll see that these 548 00:30:35,430 --> 00:30:38,190 are two heptameric rings. 549 00:30:38,190 --> 00:30:40,560 The ring that has the lid attached 550 00:30:40,560 --> 00:30:42,810 is called the cis ring. 551 00:30:42,810 --> 00:30:46,170 Or sorry-- the chamber or heptamer with the lid attached 552 00:30:46,170 --> 00:30:52,020 is cis, and the one below is trans. 553 00:30:52,020 --> 00:30:53,040 And this is huge. 554 00:30:53,040 --> 00:30:58,950 So this whole thing is on the order of 184 angstroms just 555 00:30:58,950 --> 00:31:01,780 to give some scale. 556 00:31:01,780 --> 00:31:19,700 So EL is the chaperonin, and ES is the co here. 557 00:31:19,700 --> 00:31:25,610 So what we'll do is look at the structural characteristics 558 00:31:25,610 --> 00:31:29,660 of GroEL and GroES individually and then 559 00:31:29,660 --> 00:31:30,920 think about function here. 560 00:31:40,430 --> 00:31:47,470 So for GroEL, what we have are to have to heptameric rings. 561 00:31:53,572 --> 00:32:19,540 OK, and so if we look from the top here, what we have are 562 00:32:19,540 --> 00:32:22,000 the seven subunits arranged in this ring. 563 00:32:31,150 --> 00:32:33,580 OK, and what we see is that there's 564 00:32:33,580 --> 00:32:40,670 an inner cavity that's about 45 angstroms in diameter, OK? 565 00:32:44,600 --> 00:32:52,410 And each subunit is about 60 kilodaltons, which 566 00:32:52,410 --> 00:32:55,440 is why this is called Hsp70. 567 00:32:55,440 --> 00:32:58,620 And so if we take a look in this structural depiction 568 00:32:58,620 --> 00:33:02,710 here, in the middle, this is the top view. 569 00:33:02,710 --> 00:33:05,280 OK, and the different subunits have been color-coded. 570 00:33:05,280 --> 00:33:06,900 They're all the same polypeptide. 571 00:33:06,900 --> 00:33:08,280 They're just differentiating them 572 00:33:08,280 --> 00:33:10,500 here so it's easy to see each one. 573 00:33:10,500 --> 00:33:11,820 And here's that inner cavity. 574 00:33:15,120 --> 00:33:18,377 If we look at the side view again, 575 00:33:18,377 --> 00:33:20,085 we need to consider a little more detail. 576 00:33:25,840 --> 00:33:28,170 OK, so each one of these is a 7-mer. 577 00:33:33,020 --> 00:33:36,730 OK, and each subunit of GroEL three 578 00:33:36,730 --> 00:33:43,930 domains that are organized A, I, E-- 579 00:33:43,930 --> 00:33:48,745 so apical domain, intermediate domain, and equatorial domain. 580 00:33:48,745 --> 00:33:50,620 And then if we look at this bottom ring here, 581 00:33:50,620 --> 00:33:54,350 they're organized like that. 582 00:33:54,350 --> 00:34:03,711 OK, so effectively, what we have is a back-to-back arrangement. 583 00:34:08,630 --> 00:34:16,840 OK, so we have 14 subunits and two back-to-back rings. 584 00:34:41,060 --> 00:34:47,080 And so if we take a look again at this depiction, what's 585 00:34:47,080 --> 00:34:51,340 been done is that in this top 7-mer ring, 586 00:34:51,340 --> 00:34:54,760 for one of the subunits, the three domains 587 00:34:54,760 --> 00:34:56,570 have been colored. 588 00:34:56,570 --> 00:34:58,990 OK, so we see that the apical domain 589 00:34:58,990 --> 00:35:03,760 is an orange, the intermediate domain in yellow, 590 00:35:03,760 --> 00:35:07,990 and this equatorial domain in red shown here. 591 00:35:07,990 --> 00:35:11,110 And this is one isolated subunit again with this 592 00:35:11,110 --> 00:35:13,220 color-coding here. 593 00:35:16,930 --> 00:35:24,840 So what happens when the lid binds? 594 00:35:24,840 --> 00:35:28,920 So we're currently looking at this as just GroEL 595 00:35:28,920 --> 00:35:29,760 the to heptamers. 596 00:35:29,760 --> 00:35:34,680 But we need to begin to think about GroEL with its lid. 597 00:35:34,680 --> 00:35:39,420 What happens when the lid binds is that there's 598 00:35:39,420 --> 00:35:40,545 a conformational change. 599 00:36:03,240 --> 00:36:10,630 OK, and so I'll just draw this, and then we'll 600 00:36:10,630 --> 00:36:12,310 look at the structure. 601 00:36:12,310 --> 00:36:17,220 OK, so imagine that this is one GroEL 602 00:36:17,220 --> 00:36:20,950 subunit of the 7-mer ring, OK? 603 00:36:20,950 --> 00:36:26,950 When GroES binds to that ring, what happens 604 00:36:26,950 --> 00:36:29,920 is that the GroEL subunits change 605 00:36:29,920 --> 00:36:31,660 from a closed conformation, which 606 00:36:31,660 --> 00:36:41,125 I'm kind of showing as closed, to something that's open. 607 00:36:47,880 --> 00:36:52,440 OK, so effectively, it's like opening up a hinge. 608 00:37:00,100 --> 00:37:02,680 OK, and so the consequence of this 609 00:37:02,680 --> 00:37:08,290 is that when the lid binds to this cis cavity, 610 00:37:08,290 --> 00:37:14,560 the size of the central cavity expands dramatically. 611 00:37:14,560 --> 00:37:16,810 So it basically doubles. 612 00:37:16,810 --> 00:37:20,740 And that's something that's not clearly indicated here. 613 00:37:23,590 --> 00:37:24,880 So we can modify the cartoon. 614 00:37:53,560 --> 00:37:59,460 OK, so let's take a look and then talk 615 00:37:59,460 --> 00:38:01,450 about why that's important. 616 00:38:01,450 --> 00:38:10,590 So here are two depictions where we have GroEL/GroES, 617 00:38:10,590 --> 00:38:13,680 and we can look at a GroEL subunit that 618 00:38:13,680 --> 00:38:17,360 does not have GroES bound, so with the trans ring. 619 00:38:17,360 --> 00:38:20,160 Or we can look at a GroEL subunit 620 00:38:20,160 --> 00:38:23,620 where the GroES lid is bound, so in the cis. 621 00:38:23,620 --> 00:38:26,640 OK, and so this is actual structural depiction of what 622 00:38:26,640 --> 00:38:30,840 I tried to indicate on the board here where we have closed 623 00:38:30,840 --> 00:38:31,770 and open. 624 00:38:31,770 --> 00:38:36,480 And so this opening is making this cis ring much larger 625 00:38:36,480 --> 00:38:38,820 in terms of its central cavity, OK? 626 00:38:38,820 --> 00:38:42,150 So these are major conformational changes 627 00:38:42,150 --> 00:38:45,120 and details of which are described here. 628 00:38:45,120 --> 00:38:48,360 But effectively, the two points to keep in mind 629 00:38:48,360 --> 00:38:53,328 is, one, that diameter and size of this central cavity doubles. 630 00:38:53,328 --> 00:38:54,870 And we can think about why that might 631 00:38:54,870 --> 00:38:57,690 be important in terms of accommodating a larger 632 00:38:57,690 --> 00:39:02,820 polypeptide as we get towards the functional cycle of this. 633 00:39:02,820 --> 00:39:06,450 And also, what we'll see as we move forward 634 00:39:06,450 --> 00:39:10,680 is that the distribution of hydrophobic and hydrophilic 635 00:39:10,680 --> 00:39:12,990 residues on the interior of this cavity 636 00:39:12,990 --> 00:39:19,320 changes dramatically when GroES binds here. 637 00:39:19,320 --> 00:39:23,340 So briefly, to look at GroES, what 638 00:39:23,340 --> 00:39:26,670 does that look like from a structural perspective? 639 00:39:26,670 --> 00:39:32,690 So GroES is also a heptamer. 640 00:39:45,250 --> 00:39:49,000 OK, each subunit is only about 10 kilodaltons. 641 00:39:57,211 --> 00:40:01,530 It's about 30 angstroms in height 642 00:40:01,530 --> 00:40:06,570 and about 75 angstroms across here. 643 00:40:06,570 --> 00:40:09,410 And what we see if we look at the structure 644 00:40:09,410 --> 00:40:15,170 of an individual GroES, so again, here, what we see 645 00:40:15,170 --> 00:40:19,420 is that there is a beta sheet region. 646 00:40:19,420 --> 00:40:21,110 And there is this region here that's 647 00:40:21,110 --> 00:40:23,490 described as a mobile loop. 648 00:40:23,490 --> 00:40:25,880 And if you look, the beta sheet region's on top, 649 00:40:25,880 --> 00:40:30,990 and this mobile loop is down where it binds to GroEL. 650 00:40:30,990 --> 00:40:37,250 OK, so effectively, when GroES docks onto GroEL, 651 00:40:37,250 --> 00:40:42,230 these mobile loops bind to hydrophobic peptide binding 652 00:40:42,230 --> 00:40:47,340 pockets that are on the top of this heptamer there. 653 00:40:47,340 --> 00:40:49,830 OK, here's another depiction. 654 00:40:49,830 --> 00:40:51,830 So you're seeing the beta sheet parts on top, 655 00:40:51,830 --> 00:40:55,040 and here are the mobile loops that 656 00:40:55,040 --> 00:41:00,260 can bind to peptide binding grooves of GroEL. 657 00:41:00,260 --> 00:41:06,000 AUDIENCE: So does that mean the inner cavity of the cis 658 00:41:06,000 --> 00:41:12,790 part of GroEL is always open I guess after GroES binds? 659 00:41:12,790 --> 00:41:15,320 And trans is always closed? 660 00:41:15,320 --> 00:41:19,370 Because it looks like just from the way we've drawn it. 661 00:41:19,370 --> 00:41:21,190 Does GroES bind to the other side also? 662 00:41:21,190 --> 00:41:23,750 ELIZABETH NOLAN: Yeah, so right, this is how we've drawn it. 663 00:41:23,750 --> 00:41:26,030 We've drawn it like a bullet. 664 00:41:26,030 --> 00:41:31,060 And so does GroES bind to the other side? 665 00:41:31,060 --> 00:41:34,260 And how do these two chambers function? 666 00:41:34,260 --> 00:41:36,280 OK, and as we move forward getting 667 00:41:36,280 --> 00:41:38,770 to the functional cycle, what we'll see 668 00:41:38,770 --> 00:41:41,140 is that both rings are functional, 669 00:41:41,140 --> 00:41:44,470 but they're functional at different points in the cycle. 670 00:41:44,470 --> 00:41:48,730 OK, so GroES, yes, can bind to either one, 671 00:41:48,730 --> 00:41:51,940 but it's this bullet type of shape that 672 00:41:51,940 --> 00:41:53,900 is considered to be functional. 673 00:41:53,900 --> 00:41:57,040 So you might ask, what about a football? 674 00:41:57,040 --> 00:41:59,210 If we stick another GroES on the bottom, 675 00:41:59,210 --> 00:42:01,720 we get a football-shaped species. 676 00:42:01,720 --> 00:42:03,880 And there are some in vitro studies 677 00:42:03,880 --> 00:42:08,410 that show a formation of a football with two GroEL rings 678 00:42:08,410 --> 00:42:13,090 and two GroES rings, but those are found at very high ATP 679 00:42:13,090 --> 00:42:15,230 concentrations. 680 00:42:15,230 --> 00:42:19,630 And so it's thought that they may not be significant, 681 00:42:19,630 --> 00:42:21,670 that they're a transient species effectively 682 00:42:21,670 --> 00:42:24,190 of unknown significance that at least in the test tube, 683 00:42:24,190 --> 00:42:26,860 you can form at very high ATP. 684 00:42:26,860 --> 00:42:28,170 OK, yeah? 685 00:42:28,170 --> 00:42:31,750 AUDIENCE: And then is the cis and trans, it's not predefined, 686 00:42:31,750 --> 00:42:33,925 just it depends on wherever the GroES makes it? 687 00:42:33,925 --> 00:42:34,883 ELIZABETH NOLAN: Right. 688 00:42:34,883 --> 00:42:37,210 It's going to depend on wherever the GroES is. 689 00:42:37,210 --> 00:42:39,250 OK, and what we'll see as we move forward 690 00:42:39,250 --> 00:42:42,610 is we need to think about also how ATP binds. 691 00:42:42,610 --> 00:42:49,000 And ATP binding will also happen in one or the other, 692 00:42:49,000 --> 00:42:52,600 depending at the point in the functional cycle here, OK? 693 00:42:52,600 --> 00:42:55,600 So we just want to get the structural aspects 694 00:42:55,600 --> 00:42:58,840 under control before we look at the functional cycle. 695 00:42:58,840 --> 00:43:03,770 So this is one last slide on the structure. 696 00:43:03,770 --> 00:43:09,050 And so I find this to be a really beautiful machine. 697 00:43:09,050 --> 00:43:13,420 Here we have the bullet-shaped two GroELs and one GroES. 698 00:43:13,420 --> 00:43:18,990 And here we have the different domains colored. 699 00:43:18,990 --> 00:43:21,670 And here what we have is a cutaway view 700 00:43:21,670 --> 00:43:25,540 to look at the interior of the chambers. 701 00:43:25,540 --> 00:43:28,600 And so we have the cis chamber on top, 702 00:43:28,600 --> 00:43:30,880 the trans chamber on bottom. 703 00:43:30,880 --> 00:43:35,170 And in the color-coding here for this cross-section, what 704 00:43:35,170 --> 00:43:38,860 we have in yellow are hydrophobic residues, 705 00:43:38,860 --> 00:43:42,010 and in cyan, hydrophilic residues. 706 00:43:42,010 --> 00:43:44,410 OK, and so what's important to do 707 00:43:44,410 --> 00:43:49,000 is take a look at the cis chamber and the trans chamber 708 00:43:49,000 --> 00:43:52,060 and ask, what's going on in the interior? 709 00:43:52,060 --> 00:43:54,250 And why might that be important? 710 00:43:54,250 --> 00:43:57,010 So what do we see comparing the distribution 711 00:43:57,010 --> 00:44:00,220 of yellow and cyan, or hydrophobic and hydrophilic? 712 00:44:03,900 --> 00:44:04,400 Lindsay? 713 00:44:04,400 --> 00:44:05,900 AUDIENCE: It's much more hydrophobic 714 00:44:05,900 --> 00:44:07,432 in the trans chamber. 715 00:44:07,432 --> 00:44:08,640 ELIZABETH NOLAN: Yeah, right. 716 00:44:08,640 --> 00:44:11,040 The trans chamber is much more hydrophobic 717 00:44:11,040 --> 00:44:15,120 in terms of that lining than the cis. 718 00:44:15,120 --> 00:44:20,790 So the cis chamber, as we'll see in a minute, 719 00:44:20,790 --> 00:44:23,490 is where the polypeptide will be folding. 720 00:44:23,490 --> 00:44:26,610 So a polypeptide will end up in the cis chamber, 721 00:44:26,610 --> 00:44:28,020 and the lid will be on top. 722 00:44:28,020 --> 00:44:31,440 So why might this be an important feature-- 723 00:44:31,440 --> 00:44:34,350 not only that we need this cavity size to grow, 724 00:44:34,350 --> 00:44:37,247 but we need a change in the lining to be more hydrophilic? 725 00:44:48,677 --> 00:44:50,510 AUDIENCE: Because if it's assisting folding, 726 00:44:50,510 --> 00:44:52,687 it's likely that the hydrophobic residues are 727 00:44:52,687 --> 00:44:54,270 more likely to be buried in the center 728 00:44:54,270 --> 00:44:56,210 the protein in the polypeptide. 729 00:44:56,210 --> 00:44:58,470 So you'd want to facilitate favoring 730 00:44:58,470 --> 00:45:00,250 the hydrophilic residues to be interacting 731 00:45:00,250 --> 00:45:01,880 on the outside of the protein? 732 00:45:01,880 --> 00:45:04,422 ELIZABETH NOLAN: Yeah, so often, that's right to think about. 733 00:45:04,422 --> 00:45:06,650 Where do we find different types of residues, 734 00:45:06,650 --> 00:45:09,200 say, in a protein with a complex fold? 735 00:45:09,200 --> 00:45:12,020 And typically, we think about hydrophobic residues 736 00:45:12,020 --> 00:45:15,950 on the interior and hydrophilic residues on the exterior. 737 00:45:15,950 --> 00:45:17,870 So for instance, there is a model 738 00:45:17,870 --> 00:45:20,060 of folding called hydrophobic collapse. 739 00:45:20,060 --> 00:45:23,360 And effectively, you have hydrophobic interactions, 740 00:45:23,360 --> 00:45:26,207 and then the rest of folding occurs, right? 741 00:45:26,207 --> 00:45:28,040 So you'd imagine there's a benefit to having 742 00:45:28,040 --> 00:45:29,990 a hydrophilic exterior if you want 743 00:45:29,990 --> 00:45:35,100 the exterior of the protein to be hydrophilic here. 744 00:45:35,100 --> 00:45:39,290 So what is the functional cycle? 745 00:45:39,290 --> 00:45:44,720 And in thinking about this, we need 746 00:45:44,720 --> 00:45:49,970 to think about ATPs and ATP hydrolysis. 747 00:45:52,890 --> 00:45:56,870 And what you'll find as you read is that often the model is 748 00:45:56,870 --> 00:45:59,660 drawn a bit differently depending on the paper 749 00:45:59,660 --> 00:46:00,710 you read. 750 00:46:00,710 --> 00:46:03,260 And that's because they're just some uncertainties out there. 751 00:46:03,260 --> 00:46:04,940 So don't get hung up on that. 752 00:46:04,940 --> 00:46:09,290 I have two different examples within the lecture slides here. 753 00:46:09,290 --> 00:46:11,555 OK, but if we just think about the functional cycle-- 754 00:46:21,940 --> 00:46:25,010 and I'll just draw a little bit, and then we'll go to the board. 755 00:46:25,010 --> 00:46:30,440 So imagine we have one GroEL here, OK? 756 00:46:30,440 --> 00:46:34,580 And as drawn here, there's no GroES. 757 00:46:34,580 --> 00:46:38,780 There's no peptide, and there's no ATP. 758 00:46:38,780 --> 00:46:41,630 So imagine some peptide comes in that 759 00:46:41,630 --> 00:46:44,795 needs to be folded by this machinery and ATP. 760 00:46:48,780 --> 00:46:52,610 OK, and these end up inside of the chamber. 761 00:47:01,320 --> 00:47:02,910 And then we can have our lid come in. 762 00:47:08,010 --> 00:47:15,030 OK, so now this polypeptide is in this protected cavity, 763 00:47:15,030 --> 00:47:20,880 and ATP can bind in the equatorial domain of GroEL. 764 00:47:20,880 --> 00:47:25,140 So each GroEL monomer will bind one ATP. 765 00:47:25,140 --> 00:47:28,470 So there's seven ATP bound in one heptamer 766 00:47:28,470 --> 00:47:31,464 if it's in the ATP-bound form, OK? 767 00:47:34,810 --> 00:47:39,620 And so let's take a look in a little more detail. 768 00:47:39,620 --> 00:47:41,890 So what do we see? 769 00:47:41,890 --> 00:47:44,570 And the thing to keep in mind, as I said before, 770 00:47:44,570 --> 00:47:47,560 is that both chambers are active and functional. 771 00:47:47,560 --> 00:47:49,450 They're just functional at different points 772 00:47:49,450 --> 00:47:51,880 within this overall cycle. 773 00:47:51,880 --> 00:47:56,530 OK, so if we begin here, what do we see? 774 00:47:56,530 --> 00:48:02,260 This top GroEL heptamer has no cap. 775 00:48:02,260 --> 00:48:06,760 What we see here is that we have the bottom GroEL bound 776 00:48:06,760 --> 00:48:08,800 to ADP and GroES. 777 00:48:08,800 --> 00:48:12,130 Some unfolded polypeptide comes along. 778 00:48:12,130 --> 00:48:15,910 It binds, so maybe there's some hydrophobic interaction 779 00:48:15,910 --> 00:48:18,310 between the top of GroEL and some region 780 00:48:18,310 --> 00:48:20,350 of this polypeptide. 781 00:48:20,350 --> 00:48:22,690 What do we see happening? 782 00:48:22,690 --> 00:48:26,980 The ATPs come in, so I indicated them together, there's 783 00:48:26,980 --> 00:48:29,830 some timing where there's questions. 784 00:48:29,830 --> 00:48:33,490 These ADPs from the bottom chamber are ejected. 785 00:48:33,490 --> 00:48:35,980 We see ATP binding, so there's seven-- 786 00:48:35,980 --> 00:48:36,970 one per subunit. 787 00:48:36,970 --> 00:48:40,260 The polypeptide binds, and here comes GroES. 788 00:48:40,260 --> 00:48:45,760 OK, and so once this polypeptide is 789 00:48:45,760 --> 00:48:49,990 encapsulated in this chamber, there's some residency time. 790 00:48:49,990 --> 00:48:53,600 And this is often quoted on the order of 10 seconds. 791 00:48:53,600 --> 00:48:54,917 Also note here. 792 00:48:54,917 --> 00:48:56,500 Look what happened at the bottom ring. 793 00:48:56,500 --> 00:48:58,300 GroES got ejected. 794 00:48:58,300 --> 00:49:00,880 OK, so with GroES binding here, there 795 00:49:00,880 --> 00:49:05,650 was ejection of GroES from the bottom and loss of these ADPs. 796 00:49:05,650 --> 00:49:09,180 OK, there's ATP hydrolysis during this time. 797 00:49:09,180 --> 00:49:12,610 The polypeptide is trying to find its fold. 798 00:49:12,610 --> 00:49:14,830 And then look what happens here. 799 00:49:14,830 --> 00:49:18,190 We see GroES coming into the bottom. 800 00:49:18,190 --> 00:49:20,980 Again, we have release of ADPs, release of GroES, 801 00:49:20,980 --> 00:49:24,130 and this polypeptide kicked out, which may or may not 802 00:49:24,130 --> 00:49:27,790 be in its native fold, OK? 803 00:49:27,790 --> 00:49:33,950 If we take a look showing this as a complete cycle here-- 804 00:49:33,950 --> 00:49:37,450 and again, I said before there can be some differences 805 00:49:37,450 --> 00:49:39,490 from depiction to depiction-- 806 00:49:39,490 --> 00:49:42,040 but here, we are seeing GroEL. 807 00:49:42,040 --> 00:49:45,670 We have the top one and the bottom heptamer. 808 00:49:45,670 --> 00:49:48,100 Here's some polypeptide that needs to be folded. 809 00:49:48,100 --> 00:49:53,380 It's initially grabbed by the top part of GroEL. 810 00:49:53,380 --> 00:49:54,970 ATP comes in. 811 00:49:54,970 --> 00:49:57,250 We have this ATP-bound form. 812 00:49:57,250 --> 00:49:58,630 Here comes GroES. 813 00:49:58,630 --> 00:50:01,300 The polypeptide gets pushed into this chamber, 814 00:50:01,300 --> 00:50:03,520 and now it's closed. 815 00:50:03,520 --> 00:50:07,000 We have ATPase activity, so ATP hydrolysis 816 00:50:07,000 --> 00:50:10,270 to give the ADP-bound form. 817 00:50:10,270 --> 00:50:12,890 OK, and then what happens here? 818 00:50:12,890 --> 00:50:15,400 OK, what we're seeing now, this bottom ring 819 00:50:15,400 --> 00:50:16,570 is becoming functional. 820 00:50:16,570 --> 00:50:19,690 ATP binds another polypeptide. 821 00:50:19,690 --> 00:50:24,040 OK, and then we have release of GroES in the polypeptide 822 00:50:24,040 --> 00:50:25,750 from the top chamber. 823 00:50:25,750 --> 00:50:28,330 OK, and then you can flip this and work around the cycle 824 00:50:28,330 --> 00:50:29,710 again. 825 00:50:29,710 --> 00:50:32,650 OK, so this is a case where we can 826 00:50:32,650 --> 00:50:35,470 think about the affinities of the ATP 827 00:50:35,470 --> 00:50:41,200 and the ADP-bound forms of GroEL and what that 828 00:50:41,200 --> 00:50:44,410 means GroES binding here, OK? 829 00:50:44,410 --> 00:50:46,960 And so the ADP-bound form of GroEL 830 00:50:46,960 --> 00:50:50,980 has a lower affinity for GroES than the ATP-bound form 831 00:50:50,980 --> 00:50:53,380 here for that. 832 00:50:53,380 --> 00:50:57,910 So each GroEL heptamer acts as a single functional unit, 833 00:50:57,910 --> 00:51:00,850 and both rings are active as shown here 834 00:51:00,850 --> 00:51:03,040 but in different points of the cycle. 835 00:51:03,040 --> 00:51:07,480 OK, and so the thinking is that ATP binding and hydrolysis 836 00:51:07,480 --> 00:51:11,110 drives uni-directional progression through this cycle. 837 00:51:11,110 --> 00:51:14,650 With that said, there's a lot of questions as to how. 838 00:51:14,650 --> 00:51:18,310 So what is it about this ATP binding and hydrolysis event 839 00:51:18,310 --> 00:51:23,620 that allows this work to happen? 840 00:51:23,620 --> 00:51:27,790 That's a question that I see as still pretty open. 841 00:51:27,790 --> 00:51:31,030 And so I'll close with that here now. 842 00:51:31,030 --> 00:51:33,820 I suggest to review this cycle before next time. 843 00:51:33,820 --> 00:51:37,600 And what we'll address on Wednesday 844 00:51:37,600 --> 00:51:39,610 is experiments that have been done 845 00:51:39,610 --> 00:51:42,910 to sort out what are the polypeptide substrates 846 00:51:42,910 --> 00:51:44,350 for GroEL/GroES. 847 00:51:44,350 --> 00:51:47,650 So we know they must be some important players given 848 00:51:47,650 --> 00:51:50,080 that these are essential for viability. 849 00:51:50,080 --> 00:51:50,920 What are they? 850 00:51:50,920 --> 00:51:53,670 And how is that determined?