1 00:00:00,000 --> 00:00:03,270 The following content is provided under a Creative 2 00:00:03,270 --> 00:00:04,630 Commons license. 3 00:00:04,630 --> 00:00:07,140 Your support will help MIT OpenCourseWare 4 00:00:07,140 --> 00:00:11,440 continue to offer high-quality educational resources for free. 5 00:00:11,440 --> 00:00:14,100 To make a donation or view additional materials 6 00:00:14,100 --> 00:00:17,930 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,930 --> 00:00:20,575 at ocw.mit.edu. 8 00:00:20,575 --> 00:00:22,450 ELIZABETH NOLAN: What we're going to do today 9 00:00:22,450 --> 00:00:25,170 is just discuss a few aspects of cross linking. 10 00:00:25,170 --> 00:00:27,390 So we decided it was important to introduce 11 00:00:27,390 --> 00:00:31,170 this within recitations this year, because cross-linking 12 00:00:31,170 --> 00:00:33,910 comes up time and time again. 13 00:00:33,910 --> 00:00:35,970 And there's different ways to do this, 14 00:00:35,970 --> 00:00:38,860 and different strengths and limitations 15 00:00:38,860 --> 00:00:40,810 to different approaches. 16 00:00:40,810 --> 00:00:44,280 So I guess in just thinking about this, 17 00:00:44,280 --> 00:00:47,370 what is cross-linking? 18 00:00:47,370 --> 00:00:48,840 So if you say, oh, I'm going to use 19 00:00:48,840 --> 00:00:50,500 a cross-linker for my experiment, 20 00:00:50,500 --> 00:00:51,720 what does that mean? 21 00:00:55,438 --> 00:00:56,938 AUDIENCE: Forming a covalent linkage 22 00:00:56,938 --> 00:00:59,964 between two molecules of study. 23 00:00:59,964 --> 00:01:00,880 ELIZABETH NOLAN: Yeah. 24 00:01:00,880 --> 00:01:02,980 So there's going to be formation of some sort 25 00:01:02,980 --> 00:01:06,891 of covalent linkage between two or maybe more-- 26 00:01:06,891 --> 00:01:07,390 right? 27 00:01:07,390 --> 00:01:08,848 Because some cross-linkers can have 28 00:01:08,848 --> 00:01:13,300 more than two reactive groups, OK, of study, right? 29 00:01:13,300 --> 00:01:16,420 So we're chemically joining two or more molecules. 30 00:01:16,420 --> 00:01:18,670 So why might we want to do this? 31 00:01:18,670 --> 00:01:23,054 What are possible applications? 32 00:01:23,054 --> 00:01:25,119 AUDIENCE: Study protein-protein interactions. 33 00:01:25,119 --> 00:01:26,410 ELIZABETH NOLAN: So that's one. 34 00:01:26,410 --> 00:01:28,450 So protein-protein interactions, right. 35 00:01:28,450 --> 00:01:32,200 And that could be identifying unknown protein-protein 36 00:01:32,200 --> 00:01:36,850 interactions or maybe you know two proteins interact, act 37 00:01:36,850 --> 00:01:38,740 but you don't know how, right? 38 00:01:38,740 --> 00:01:40,930 And you decide to use cross-linking 39 00:01:40,930 --> 00:01:42,780 as a way to probe that. 40 00:01:42,780 --> 00:01:46,030 So how might cross-linking help with studying a known 41 00:01:46,030 --> 00:01:47,905 protein-protein interaction? 42 00:01:50,880 --> 00:01:52,930 AUDIENCE: Start getting an idea of where 43 00:01:52,930 --> 00:01:56,350 the proteins are actually interacting or which residues 44 00:01:56,350 --> 00:01:58,580 [INAUDIBLE] 45 00:01:58,580 --> 00:01:59,510 ELIZABETH NOLAN: Yeah. 46 00:01:59,510 --> 00:02:02,648 AUDIENCE: It could allow you to isolate them. 47 00:02:02,648 --> 00:02:09,162 [INAUDIBLE] 48 00:02:09,162 --> 00:02:10,120 ELIZABETH NOLAN: Right. 49 00:02:10,120 --> 00:02:13,400 So maybe there's an unknown one, and you fish that out, 50 00:02:13,400 --> 00:02:16,280 because a cross-linker was used, right? 51 00:02:16,280 --> 00:02:17,720 And you know what one of them are. 52 00:02:17,720 --> 00:02:23,360 Or maybe, say, we know that these two proteins interact 53 00:02:23,360 --> 00:02:26,320 somehow, but we don't know how. 54 00:02:26,320 --> 00:02:35,150 So is it on an interface on this side versus maybe 55 00:02:35,150 --> 00:02:43,440 the other side versus maybe behind the board, et cetera. 56 00:02:43,440 --> 00:02:45,290 And so, there's many ways to study 57 00:02:45,290 --> 00:02:46,970 protein-protein interaction. 58 00:02:46,970 --> 00:02:49,070 And really, how I'll present cross-linking today 59 00:02:49,070 --> 00:02:52,950 is in the context of this particular application, 60 00:02:52,950 --> 00:02:54,230 but there are many others. 61 00:02:54,230 --> 00:02:55,813 But if we just think, we've seen a lot 62 00:02:55,813 --> 00:02:58,580 of protein-protein interactions in this course, right? 63 00:02:58,580 --> 00:03:02,060 So just even today, ClpXP is an example, right? 64 00:03:02,060 --> 00:03:04,340 We saw protein nucleotide interaction 65 00:03:04,340 --> 00:03:08,030 with the ribosome GroEL GroES is an example 66 00:03:08,030 --> 00:03:10,002 of protein-protein interactions, right? 67 00:03:10,002 --> 00:03:11,960 And they've been studied by many other methods, 68 00:03:11,960 --> 00:03:14,270 like crystallography for instance. 69 00:03:14,270 --> 00:03:17,240 But sometimes maybe it's not possible to get a structure, 70 00:03:17,240 --> 00:03:18,420 right? 71 00:03:18,420 --> 00:03:21,050 And you want to define an interaction surface 72 00:03:21,050 --> 00:03:25,250 or know exactly what residues are important. 73 00:03:25,250 --> 00:03:29,080 So here, say, is protein-protein. 74 00:03:35,814 --> 00:03:39,630 But that could be generalized to any other type of molecule, 75 00:03:39,630 --> 00:03:41,940 like RNA, DNA, right? 76 00:03:41,940 --> 00:03:44,550 What about a single protein? 77 00:03:44,550 --> 00:03:46,890 So can you use cross-linking to learn more 78 00:03:46,890 --> 00:03:50,610 about tertiary structure, quaternary structure? 79 00:03:50,610 --> 00:03:54,960 So imagine for instance, rather than two separate proteins, 80 00:03:54,960 --> 00:03:59,730 we have one protein where there's some flexible linker. 81 00:04:04,440 --> 00:04:07,350 And we have reason to believe these different domains 82 00:04:07,350 --> 00:04:08,590 interact. 83 00:04:08,590 --> 00:04:09,730 But how do they interact? 84 00:04:09,730 --> 00:04:14,700 Again, is it something like this undergoes 85 00:04:14,700 --> 00:04:16,380 some conformational change and they're 86 00:04:16,380 --> 00:04:22,670 like this versus other possibilities here? 87 00:04:22,670 --> 00:04:24,810 So what about just other applications 88 00:04:24,810 --> 00:04:27,540 of cross-linking chemistry before we 89 00:04:27,540 --> 00:04:30,750 look at some examples of molecules? 90 00:04:30,750 --> 00:04:34,920 So we can capture and identify binding partners, 91 00:04:34,920 --> 00:04:36,300 as Lindsey indicated. 92 00:04:36,300 --> 00:04:40,350 We can study known interactions. 93 00:04:40,350 --> 00:04:43,930 Where else could this come up? 94 00:04:43,930 --> 00:04:45,990 While it wasn't defined in this way, 95 00:04:45,990 --> 00:04:47,880 we've seen certain technology that 96 00:04:47,880 --> 00:04:51,298 takes advantage of cross-linking chemistry often. 97 00:04:51,298 --> 00:04:53,637 AUDIENCE: Within the realm of biological things, 98 00:04:53,637 --> 00:04:54,262 it's used for-- 99 00:04:54,262 --> 00:04:57,720 I mean, if you want to find a functional root. 100 00:04:57,720 --> 00:05:00,530 So like bioaccumulation or general bioconjugate chemistry 101 00:05:00,530 --> 00:05:02,132 for [INAUDIBLE] 102 00:05:02,132 --> 00:05:03,090 ELIZABETH NOLAN: Right. 103 00:05:03,090 --> 00:05:04,530 So general. 104 00:05:04,530 --> 00:05:08,580 Exactly, general bioconjugate or conjugation chemistry. 105 00:05:08,580 --> 00:05:12,180 So maybe you want to attach a tag to a purified protein. 106 00:05:12,180 --> 00:05:15,000 Maybe you want to modify an antibody. 107 00:05:15,000 --> 00:05:17,140 Similar chemistry can be employed. 108 00:05:17,140 --> 00:05:20,490 And likewise, even like from application standpoint, 109 00:05:20,490 --> 00:05:21,450 a mobilization. 110 00:05:21,450 --> 00:05:23,850 So say you need to make your own resin 111 00:05:23,850 --> 00:05:26,100 to do some sort of affinity chromatography 112 00:05:26,100 --> 00:05:29,700 and you want to attach a protein or an antibody to that, 113 00:05:29,700 --> 00:05:33,827 you can use the types of chemistry shown here. 114 00:05:33,827 --> 00:05:35,910 So we're going to talk about a few different types 115 00:05:35,910 --> 00:05:41,340 of cross-linker and the chemistry, and pros and cons. 116 00:05:41,340 --> 00:05:48,120 And just as a general overview, I'll describe types. 117 00:05:56,700 --> 00:06:00,650 So we just heard the word homobifunctional. 118 00:06:00,650 --> 00:06:10,260 So homobifunctional versus heterobifunctional. 119 00:06:16,870 --> 00:06:17,370 OK. 120 00:06:17,370 --> 00:06:19,740 And this refers to the reactive groups. 121 00:06:19,740 --> 00:06:22,410 So we need to talk about what types of chemistry 122 00:06:22,410 --> 00:06:25,320 is going to be used to do cross-linking. 123 00:06:25,320 --> 00:06:30,015 So this refers to reactive groups. 124 00:06:34,540 --> 00:06:44,160 And then another classification will be non-specific 125 00:06:44,160 --> 00:06:46,230 versus specific. 126 00:06:46,230 --> 00:06:50,490 And so, this doesn't refer to, say, 127 00:06:50,490 --> 00:06:52,650 the chemical reaction between the cross-linker 128 00:06:52,650 --> 00:06:56,340 and whatever it's hitting, but rather whether or not 129 00:06:56,340 --> 00:06:58,710 the cross-linking reagent is site-specifically 130 00:06:58,710 --> 00:07:03,180 attached to a protein or biomolecule of interest or not. 131 00:07:40,470 --> 00:07:44,600 If we just think about this non-specific versus specific, 132 00:07:44,600 --> 00:07:47,660 if we want to attach a cross-linker 133 00:07:47,660 --> 00:07:53,021 at some specific site in a protein, how can we do that? 134 00:07:53,021 --> 00:07:56,240 So think back to the ribosome discussion, 135 00:07:56,240 --> 00:08:01,280 where unnatural amino acid incorporation was not attached, 136 00:08:01,280 --> 00:08:03,560 but was introduced. 137 00:08:03,560 --> 00:08:05,090 So that's one possibility. 138 00:08:05,090 --> 00:08:10,250 If you have an amino azyl tRNA synthetase and a tRNA that 139 00:08:10,250 --> 00:08:12,440 can allow some sort of cross-linker 140 00:08:12,440 --> 00:08:14,810 to be introduced site-specifically, 141 00:08:14,810 --> 00:08:17,720 and it works for your experimental situation, 142 00:08:17,720 --> 00:08:19,670 you can do that. 143 00:08:19,670 --> 00:08:22,520 So we saw benzophenone, which is a cross-linker 144 00:08:22,520 --> 00:08:26,930 and the evolution of that orthogonal ribosome ribo-x. 145 00:08:26,930 --> 00:08:29,000 But let's say you can't do that, right? 146 00:08:29,000 --> 00:08:30,840 So for instance as far as I know, 147 00:08:30,840 --> 00:08:36,470 there's no tRNA AARS pair for benzophenone 148 00:08:36,470 --> 00:08:38,539 in a eukaryotic cell, right? 149 00:08:38,539 --> 00:08:40,510 Or maybe in some circumstance. 150 00:08:40,510 --> 00:08:43,400 What is something just using standard biochemistry 151 00:08:43,400 --> 00:08:46,470 you could do? 152 00:08:46,470 --> 00:08:50,055 So what type of residues can be modified in a protein? 153 00:08:55,708 --> 00:08:56,754 AUDIENCE: Cysteine. 154 00:08:56,754 --> 00:08:57,670 ELIZABETH NOLAN: Yeah. 155 00:08:57,670 --> 00:08:58,990 So cysteine, lysine. 156 00:08:58,990 --> 00:09:02,350 These are common side chains that are modified. 157 00:09:02,350 --> 00:09:04,570 And what would you say is more typically employed 158 00:09:04,570 --> 00:09:08,350 if you want to introduce a site-specific modification 159 00:09:08,350 --> 00:09:10,230 using chemistry? 160 00:09:10,230 --> 00:09:11,180 AUDIENCE: Cysteine. 161 00:09:11,180 --> 00:09:12,730 ELIZABETH NOLAN: Cysteine, right? 162 00:09:12,730 --> 00:09:15,940 So if you have an individual cysteine that's in the protein 163 00:09:15,940 --> 00:09:19,150 or maybe you use site-directed mutagenesis, 164 00:09:19,150 --> 00:09:21,250 you know where that cysteine is, and then you 165 00:09:21,250 --> 00:09:24,130 can modify it with some reagent there. 166 00:09:24,130 --> 00:09:26,180 We'll come back to that in a minute. 167 00:09:26,180 --> 00:09:30,040 So in terms of reactive groups then on the protein, 168 00:09:30,040 --> 00:09:32,140 we can think about lysines, right? 169 00:09:32,140 --> 00:09:36,340 We have the epsilon amino group, cysteines. 170 00:09:36,340 --> 00:09:38,650 We have the thiol. 171 00:09:38,650 --> 00:09:41,080 What do we need to think about for our chemistry 172 00:09:41,080 --> 00:09:43,060 when thinking about these types of side chains 173 00:09:43,060 --> 00:09:46,830 and wanting to do a reaction? 174 00:09:46,830 --> 00:09:50,870 So under what conditions do we have a good nucleophile? 175 00:09:50,870 --> 00:09:51,370 Pardon? 176 00:09:51,370 --> 00:09:52,340 AUDIENCE: [INAUDIBLE] 177 00:09:52,340 --> 00:09:53,256 ELIZABETH NOLAN: Yeah. 178 00:09:53,256 --> 00:09:55,480 So we need to think about the basicity, right? 179 00:09:55,480 --> 00:09:58,880 The PKA of these groups, right? 180 00:09:58,880 --> 00:10:01,720 That's very key here for that. 181 00:10:04,820 --> 00:10:07,890 What else do we need to think about? 182 00:10:07,890 --> 00:10:12,290 What other factors might govern reactivity, just thinking 183 00:10:12,290 --> 00:10:12,790 broadly? 184 00:10:12,790 --> 00:10:15,040 So PKA. 185 00:10:15,040 --> 00:10:18,040 For your amine, it will be type of amine. 186 00:10:18,040 --> 00:10:20,820 For a cysteine, redox will play a role, right? 187 00:10:20,820 --> 00:10:22,960 You can't have your cysteine and a disulfide. 188 00:10:22,960 --> 00:10:26,290 It needs to be the free thiol form. 189 00:10:26,290 --> 00:10:28,060 So these are all things to keep in mind. 190 00:10:31,000 --> 00:10:35,510 So Alex has used a homobifunctional cross-linker. 191 00:10:35,510 --> 00:10:39,874 Why did you use a homobifunctional cross-linker? 192 00:10:39,874 --> 00:10:43,770 AUDIENCE: It was to stabilize a nanoparticle. 193 00:10:43,770 --> 00:10:46,400 ELIZABETH NOLAN: To stabilize a nanoparticle. 194 00:10:46,400 --> 00:10:46,900 OK. 195 00:10:46,900 --> 00:10:49,022 So very different type of application here. 196 00:10:49,022 --> 00:10:50,980 AUDIENCE: Yeah, that's why I didn't mention it. 197 00:10:50,980 --> 00:10:53,600 ELIZABETH NOLAN: That's fine. 198 00:10:53,600 --> 00:10:54,100 Yeah. 199 00:10:54,100 --> 00:10:56,950 We're not doing much with nanoparticles here. 200 00:10:56,950 --> 00:11:02,150 But let's say we want to use a non-specific homobifunction. 201 00:11:02,150 --> 00:11:05,140 So this was non-specific cross-linker 202 00:11:05,140 --> 00:11:08,570 to look at some protein-protein interaction, right? 203 00:11:08,570 --> 00:11:17,070 So if we just suppose, for instance, we have some protein 204 00:11:17,070 --> 00:11:22,520 A and we think it interacts somehow with protein B, 205 00:11:22,520 --> 00:11:26,680 how can we use cross-linkers to study this? 206 00:11:26,680 --> 00:11:29,100 So let's take a look at an example 207 00:11:29,100 --> 00:11:32,280 of a homobifunctional cross-linker in terms 208 00:11:32,280 --> 00:11:34,095 of design. 209 00:11:55,092 --> 00:11:57,020 So this one will be amine reactive. 210 00:12:21,110 --> 00:12:23,590 And its name is DSS here. 211 00:12:27,640 --> 00:12:30,640 So effectively, if we want to dissect 212 00:12:30,640 --> 00:12:34,450 this structure into different components, what do we have? 213 00:12:41,198 --> 00:12:44,416 AUDIENCE: Two leaving groups kind of linking. 214 00:12:44,416 --> 00:12:46,415 ELIZABETH NOLAN: So we have two reactive groups, 215 00:12:46,415 --> 00:12:49,310 or leaving group, separated by a linker. 216 00:12:49,310 --> 00:12:53,820 And in this case, we have two NHS or 6-cinnamyl esters, 217 00:12:53,820 --> 00:12:55,070 right? 218 00:12:55,070 --> 00:12:56,375 That are amine reactive. 219 00:13:11,785 --> 00:13:15,860 So what's the product of reacting 220 00:13:15,860 --> 00:13:20,210 an alpha amino group or a lysine epsilon amino group with an NHS 221 00:13:20,210 --> 00:13:21,540 ester? 222 00:13:21,540 --> 00:13:22,990 What do we get? 223 00:13:22,990 --> 00:13:23,657 AUDIENCE: Amide. 224 00:13:23,657 --> 00:13:25,031 ELIZABETH NOLAN: An amide, right? 225 00:13:25,031 --> 00:13:25,980 We get an amide bond. 226 00:13:25,980 --> 00:13:34,340 And then we have this linker or spacer region. 227 00:13:34,340 --> 00:13:35,166 OK? 228 00:13:35,166 --> 00:13:36,060 Here. 229 00:13:36,060 --> 00:13:39,060 So two amine reactive groups and a linker, or spacer. 230 00:13:39,060 --> 00:13:43,290 And in this particular case, this linker or spacer 231 00:13:43,290 --> 00:13:47,100 is about 11 angstroms and it's flexible. 232 00:13:51,630 --> 00:13:54,990 And it's stable and cannot be cleaved. 233 00:13:59,430 --> 00:14:02,370 So in the case of Alex's project, 234 00:14:02,370 --> 00:14:05,450 this was used to stabilize a nanoparticle. 235 00:14:05,450 --> 00:14:08,760 Did you have a pure nanoparticle? 236 00:14:08,760 --> 00:14:11,630 Or was this in a very complicated mixture? 237 00:14:11,630 --> 00:14:14,130 AUDIENCE: It's very not in this course. 238 00:14:14,130 --> 00:14:16,725 ELIZABETH NOLAN: So what's going to happen if this reagent, 239 00:14:16,725 --> 00:14:18,760 say, is added to cell lysate? 240 00:14:25,290 --> 00:14:28,445 What are you going to get? 241 00:14:28,445 --> 00:14:30,940 AUDIENCE: Random cross-linking with a bunch 242 00:14:30,940 --> 00:14:34,524 of different lysate proteins [INAUDIBLE].. 243 00:14:34,524 --> 00:14:35,440 ELIZABETH NOLAN: Yeah. 244 00:14:35,440 --> 00:14:37,880 So there's a high, high likelihood 245 00:14:37,880 --> 00:14:41,990 of a lot of different cross-links, right? 246 00:14:41,990 --> 00:14:44,570 So potentially a big mess, right? 247 00:14:44,570 --> 00:14:46,010 High likelihood, right? 248 00:14:46,010 --> 00:14:50,630 Because you have no control over where these reactive groups 249 00:14:50,630 --> 00:14:52,190 are going to hit. 250 00:14:52,190 --> 00:14:55,203 And do most proteins have lysine residues? 251 00:14:55,203 --> 00:14:56,450 Yeah. 252 00:14:56,450 --> 00:14:58,826 Do all proteins have an alpha amino group? 253 00:14:58,826 --> 00:15:00,830 Yeah. 254 00:15:00,830 --> 00:15:04,010 Well, some might be modified, but anyhow. 255 00:15:04,010 --> 00:15:07,470 You have very little control with this type of reagent. 256 00:15:07,470 --> 00:15:09,410 So then the question is, if you use it, 257 00:15:09,410 --> 00:15:13,910 how are you going to fish out your desired 258 00:15:13,910 --> 00:15:15,680 protein-protein interaction? 259 00:15:15,680 --> 00:15:18,050 Or even if you're working with two purified proteins 260 00:15:18,050 --> 00:15:20,180 and they have multiple lysines, you 261 00:15:20,180 --> 00:15:22,580 can end up getting multiple cross-links, right? 262 00:15:22,580 --> 00:15:24,860 So maybe that's helpful for initially identifying 263 00:15:24,860 --> 00:15:26,700 that an interaction exists. 264 00:15:26,700 --> 00:15:29,330 But in terms of getting more detailed information in terms 265 00:15:29,330 --> 00:15:33,560 of how do these actually interact, 266 00:15:33,560 --> 00:15:35,461 that may be tough here. 267 00:15:35,461 --> 00:15:35,960 OK? 268 00:15:35,960 --> 00:15:41,950 So easy to come by, but potential complications. 269 00:15:41,950 --> 00:15:44,240 Just in terms of thinking about this in the linker, 270 00:15:44,240 --> 00:15:46,460 why is it important to think about the linker 271 00:15:46,460 --> 00:15:50,300 and your choice of some reagent here? 272 00:15:50,300 --> 00:15:52,370 So what properties does the linker give? 273 00:15:59,735 --> 00:16:01,220 AUDIENCE: [INAUDIBLE] to the link, 274 00:16:01,220 --> 00:16:04,430 then I guess its flexibility will determine 275 00:16:04,430 --> 00:16:09,144 how close the two proteins have to be in space for those to be 276 00:16:09,144 --> 00:16:09,724 [INAUDIBLE]. 277 00:16:09,724 --> 00:16:10,640 ELIZABETH NOLAN: Yeah. 278 00:16:10,640 --> 00:16:12,890 So there's some constraints imposed 279 00:16:12,890 --> 00:16:17,090 by the linker in terms of how close together or far away 280 00:16:17,090 --> 00:16:18,680 are groups that react. 281 00:16:18,680 --> 00:16:20,602 What else comes with the linker? 282 00:16:20,602 --> 00:16:22,685 How does it affect the properties of the molecule? 283 00:16:22,685 --> 00:16:24,308 Alex? 284 00:16:24,308 --> 00:16:28,430 AUDIENCE: I was going to say it can dictate how likely you 285 00:16:28,430 --> 00:16:33,870 get a cross-linking on the same molecule between two amines. 286 00:16:33,870 --> 00:16:37,086 If you make it short enough, so that it 287 00:16:37,086 --> 00:16:38,812 can't reach the next lycine or something, 288 00:16:38,812 --> 00:16:41,454 then it can prevent [INAUDIBLE] 289 00:16:41,454 --> 00:16:42,370 ELIZABETH NOLAN: Yeah. 290 00:16:42,370 --> 00:16:44,590 May be able to. 291 00:16:44,590 --> 00:16:48,169 So what's an inherent property of a molecule? 292 00:16:48,169 --> 00:16:49,710 AUDIENCE: It might affect solubility. 293 00:16:49,710 --> 00:16:50,370 ELIZABETH NOLAN: Yeah. 294 00:16:50,370 --> 00:16:50,870 Right. 295 00:16:50,870 --> 00:16:53,170 It may affect solubility. 296 00:16:53,170 --> 00:16:57,250 So linkers can be-- this is a bunch of CH2 groups, 297 00:16:57,250 --> 00:17:00,190 relatively hydrophobic, right? 298 00:17:00,190 --> 00:17:03,400 There can be more hydrophilic linkers or other strategies. 299 00:17:03,400 --> 00:17:06,130 And then the question is, does that matter? 300 00:17:06,130 --> 00:17:09,160 Does the solubility properties work 301 00:17:09,160 --> 00:17:10,900 with your experiment or not? 302 00:17:10,900 --> 00:17:14,109 But imagine if you want to do cross-linking in a live cell, 303 00:17:14,109 --> 00:17:16,540 you need that cross-linker to get into the cell. 304 00:17:16,540 --> 00:17:19,750 So you need to think about membrane permeability 305 00:17:19,750 --> 00:17:21,660 and what happens after that. 306 00:17:21,660 --> 00:17:22,160 Here. 307 00:17:22,160 --> 00:17:24,700 So the linker is another critical aspect. 308 00:17:24,700 --> 00:17:26,980 And so, if you're ever working with a cross-linker, 309 00:17:26,980 --> 00:17:30,490 that's something you want to think about in addition to what 310 00:17:30,490 --> 00:17:33,440 types of side chains or what types of biomolecules 311 00:17:33,440 --> 00:17:35,170 do you want to modify. 312 00:17:35,170 --> 00:17:39,000 So let's look at an example of a heterobifunctional linker. 313 00:17:47,960 --> 00:17:48,790 It's not linker. 314 00:17:51,100 --> 00:17:51,600 Yeah. 315 00:17:51,600 --> 00:17:53,350 Well, it is cross-linker. 316 00:17:53,350 --> 00:17:53,850 OK. 317 00:17:56,520 --> 00:18:00,400 So this one will have a different type of spacer group. 318 00:18:00,400 --> 00:18:01,910 So it will be with a cyclohexyl. 319 00:18:27,600 --> 00:18:29,125 So what do we have in this case? 320 00:18:44,820 --> 00:18:47,150 Steve? 321 00:18:47,150 --> 00:18:50,502 AUDIENCE: So you have an NHS ester and also a maleimide. 322 00:18:50,502 --> 00:18:57,502 And then the sulfonate group probably helps the solubility. 323 00:18:57,502 --> 00:18:58,460 ELIZABETH NOLAN: Right. 324 00:18:58,460 --> 00:19:00,350 So there's a bunch of interesting aspects 325 00:19:00,350 --> 00:19:01,260 to this molecule. 326 00:19:01,260 --> 00:19:06,520 So we have the NHS ester to react with an amine. 327 00:19:06,520 --> 00:19:13,795 Right here we have a maleimide, which will react with thiols. 328 00:19:22,211 --> 00:19:24,970 So heterobifunctional, because there's 329 00:19:24,970 --> 00:19:27,400 two different reactive groups for different types of side 330 00:19:27,400 --> 00:19:28,610 chains. 331 00:19:28,610 --> 00:19:32,710 And then, as Steve mentioned, we have this group here. 332 00:19:32,710 --> 00:19:35,230 And so, this is to improve water solubility. 333 00:19:40,850 --> 00:19:41,770 OK. 334 00:19:41,770 --> 00:19:44,468 And then what do we have in this linker region? 335 00:19:44,468 --> 00:19:46,560 AUDIENCE: A cyclohexyl instead of the aliphatic-- 336 00:19:46,560 --> 00:19:48,080 ELIZABETH NOLAN: Yeah. 337 00:19:48,080 --> 00:19:49,105 And what does that give? 338 00:19:53,518 --> 00:19:54,789 AUDIENCE: Isn't it rigid? 339 00:19:54,789 --> 00:19:56,080 ELIZABETH NOLAN: Yeah, exactly. 340 00:19:56,080 --> 00:19:57,340 Like cyclohexyl, right? 341 00:19:57,340 --> 00:19:59,216 Think about chair conformation, rather 342 00:19:59,216 --> 00:20:00,340 than what I have done here. 343 00:20:00,340 --> 00:20:03,450 But it will give a more rigid linker, 344 00:20:03,450 --> 00:20:06,220 and also shorter than what we see up here. 345 00:20:10,441 --> 00:20:13,234 So this is on the order of eight angstroms. 346 00:20:17,980 --> 00:20:21,080 So how might this molecule be used? 347 00:20:30,537 --> 00:20:33,310 What could you do with it that you can't do with this one? 348 00:20:38,150 --> 00:20:41,074 AUDIENCE: Cross-link cysteine and lycine [INAUDIBLE] 349 00:20:41,074 --> 00:20:41,990 ELIZABETH NOLAN: Yeah. 350 00:20:41,990 --> 00:20:43,520 Well, that's the first point, right? 351 00:20:43,520 --> 00:20:45,920 You can have two different groups. 352 00:20:45,920 --> 00:20:48,080 One end will react with a cysteine. 353 00:20:48,080 --> 00:20:51,200 One with some lysine. 354 00:20:51,200 --> 00:20:54,461 So is this specific, or non-specific, or both? 355 00:20:54,461 --> 00:20:56,210 AUDIENCE: Probably depends on the context. 356 00:20:56,210 --> 00:20:56,750 ELIZABETH NOLAN: Yeah. 357 00:20:56,750 --> 00:20:57,250 Right. 358 00:20:57,250 --> 00:20:59,270 Could depend on the context. 359 00:20:59,270 --> 00:21:02,760 And then from the standpoint of specific cross-linking-- 360 00:21:02,760 --> 00:21:05,840 which I would argue is a better use of this compound-- 361 00:21:05,840 --> 00:21:09,110 what can you do? 362 00:21:09,110 --> 00:21:12,740 Just imagine you have some protein of interest 363 00:21:12,740 --> 00:21:15,110 and maybe you want to label it here. 364 00:21:15,110 --> 00:21:17,760 And you have some side chain. 365 00:21:17,760 --> 00:21:26,450 So site-directed mutagenesis to put in a cysteine. 366 00:21:26,450 --> 00:21:31,940 And then you can modify that there, 367 00:21:31,940 --> 00:21:40,170 such that you have cross-linking reagent, right? 368 00:21:55,920 --> 00:21:59,580 And then you can imagine whatever 369 00:21:59,580 --> 00:22:01,130 your experiment is here. 370 00:22:04,539 --> 00:22:09,920 So again, thinking about using this compound in, say, 371 00:22:09,920 --> 00:22:13,400 a complicated mixture, like a cell lysate-- 372 00:22:13,400 --> 00:22:17,270 you want to see if there's any binding partners or whatever. 373 00:22:17,270 --> 00:22:20,050 What's the limitation in terms of reactivity 374 00:22:20,050 --> 00:22:26,325 of this amine group that you would use in that second step? 375 00:22:29,710 --> 00:22:30,850 Where do you lack control? 376 00:22:37,568 --> 00:22:39,056 AUDIENCE: You still can't control 377 00:22:39,056 --> 00:22:43,040 for the alpha for the N-terminal reaction, right? 378 00:22:43,040 --> 00:22:46,930 ELIZABETH NOLAN: What do you mean by that? 379 00:22:46,930 --> 00:22:48,730 AUDIENCE: So if the [INAUDIBLE] is free, 380 00:22:48,730 --> 00:22:52,114 then would you have comparable reactivity 381 00:22:52,114 --> 00:22:54,534 between the N-terminals, and, for example, your desired 382 00:22:54,534 --> 00:22:56,117 lycine? 383 00:22:56,117 --> 00:22:56,950 ELIZABETH NOLAN: OK. 384 00:22:56,950 --> 00:22:58,810 So that could be an issue. 385 00:23:01,580 --> 00:23:05,560 So do lycines and N-terminal alpha amino groups 386 00:23:05,560 --> 00:23:07,560 have different reactivity? 387 00:23:07,560 --> 00:23:09,010 Do they have different PKAs? 388 00:23:09,010 --> 00:23:11,110 And is that something you could control? 389 00:23:11,110 --> 00:23:12,130 Maybe, maybe not. 390 00:23:12,130 --> 00:23:15,400 But more broadly than that, so you 391 00:23:15,400 --> 00:23:17,920 have an issue that it will react, 392 00:23:17,920 --> 00:23:21,790 let's say, with any amine, right? 393 00:23:21,790 --> 00:23:23,515 Can you control when it reacts? 394 00:23:27,222 --> 00:23:29,602 AUDIENCE: To some extent [INAUDIBLE] pH. 395 00:23:31,820 --> 00:23:33,570 ELIZABETH NOLAN: So what are you thinking? 396 00:23:35,922 --> 00:23:36,672 AUDIENCE: If you-- 397 00:23:40,640 --> 00:23:43,310 ELIZABETH NOLAN: So if you think about just experimental design, 398 00:23:43,310 --> 00:23:43,810 right? 399 00:23:43,810 --> 00:23:47,880 And say you were to try to use pH to control reactivity-- 400 00:23:47,880 --> 00:23:52,377 and I'm defining this broadly-- reacting with any amino group. 401 00:23:52,377 --> 00:23:54,710 So we're not going to try to do something to selectively 402 00:23:54,710 --> 00:23:57,170 label one, right? 403 00:23:57,170 --> 00:24:00,820 This is reactive. 404 00:24:00,820 --> 00:24:03,130 It will react, right? 405 00:24:03,130 --> 00:24:08,060 So would pH change your whole buffer? 406 00:24:08,060 --> 00:24:10,000 Or pH change the cell lysate, and then 407 00:24:10,000 --> 00:24:11,740 switch to turn on reactivity? 408 00:24:11,740 --> 00:24:13,120 Probably not. 409 00:24:13,120 --> 00:24:14,650 Probably not, right? 410 00:24:14,650 --> 00:24:19,900 That, I'd say is not very likely. 411 00:24:19,900 --> 00:24:21,640 So the issue I'm getting at here is 412 00:24:21,640 --> 00:24:26,260 that you have little temporal control or spatial control 413 00:24:26,260 --> 00:24:28,390 of an NHS ester. 414 00:24:28,390 --> 00:24:32,200 It will react with an amine provided your conditions are 415 00:24:32,200 --> 00:24:33,230 appropriate. 416 00:24:33,230 --> 00:24:38,350 So just getting back to this pH issue and a little digression, 417 00:24:38,350 --> 00:24:43,570 if you want to use something like an NHS ester 418 00:24:43,570 --> 00:24:46,300 in a test tube experiment, what you 419 00:24:46,300 --> 00:24:47,640 need to think about beyond pH? 420 00:24:51,250 --> 00:24:53,944 So what do you need to think about with the buffer? 421 00:24:53,944 --> 00:24:57,210 AUDIENCE: You don't have something [INAUDIBLE] buffer 422 00:24:57,210 --> 00:25:01,170 [INAUDIBLE] so you might want to use the phosphate buffer, 423 00:25:01,170 --> 00:25:03,886 something that doesn't [AUDIO OUT] 424 00:25:03,886 --> 00:25:06,290 ELIZABETH NOLAN: So this is a key point. 425 00:25:06,290 --> 00:25:08,060 You need to think about cross-reactivity 426 00:25:08,060 --> 00:25:09,240 with the buffer. 427 00:25:09,240 --> 00:25:11,690 So if you have tris buffer, you have amine. 428 00:25:11,690 --> 00:25:15,410 If you have a buffer that's like glycine, there's amine, right? 429 00:25:15,410 --> 00:25:18,980 And your buffer concentration in most instances 430 00:25:18,980 --> 00:25:22,220 is much higher than whatever the concentration is 431 00:25:22,220 --> 00:25:26,450 of the molecule you want to actually modify, right? 432 00:25:26,450 --> 00:25:30,260 If you think about 10 million molar tris or 75 million 433 00:25:30,260 --> 00:25:33,530 molar tris compared to micromolar or nanomolar 434 00:25:33,530 --> 00:25:35,750 of some protein, so you need to have 435 00:25:35,750 --> 00:25:37,970 a buffer that's not reactive. 436 00:25:37,970 --> 00:25:40,610 You need to have an appropriate PKA. 437 00:25:40,610 --> 00:25:42,770 Those are important considerations. 438 00:25:42,770 --> 00:25:45,800 You need to know that your reagent is good. 439 00:25:45,800 --> 00:25:47,570 Sorry, appropriate pH. 440 00:25:47,570 --> 00:25:50,190 What about the thiol here? 441 00:25:50,190 --> 00:25:52,790 What do you need to think about if you're doing a test tube 442 00:25:52,790 --> 00:25:56,600 experiment and want to modify a thiol with a maleimide 443 00:25:56,600 --> 00:25:58,700 or something else, like iodoacetamide 444 00:25:58,700 --> 00:26:00,590 that we saw last time? 445 00:26:00,590 --> 00:26:04,370 AUDIENCE: Buffers need to avoid DDT. 446 00:26:04,370 --> 00:26:05,810 ELIZABETH NOLAN: So what's DDT? 447 00:26:05,810 --> 00:26:06,988 AUDIENCE: [INAUDIBLE] 448 00:26:06,988 --> 00:26:07,946 ELIZABETH NOLAN: Right. 449 00:26:07,946 --> 00:26:09,971 Or BME, beta mercapto ethanol. 450 00:26:09,971 --> 00:26:10,470 Right. 451 00:26:10,470 --> 00:26:14,200 Even before that step, what you need to make sure? 452 00:26:14,200 --> 00:26:17,310 So what if there's multiple cysteines? 453 00:26:20,160 --> 00:26:21,952 AUDIENCE: That they're not [INAUDIBLE].. 454 00:26:21,952 --> 00:26:22,910 ELIZABETH NOLAN: Right. 455 00:26:22,910 --> 00:26:26,630 So either inter- or intramolecular, right? 456 00:26:26,630 --> 00:26:30,080 So if a reducing agent's added and the reducing agent 457 00:26:30,080 --> 00:26:32,050 is thiol-based, again, you're going 458 00:26:32,050 --> 00:26:35,220 to have much more reducing agent than your protein of interest, 459 00:26:35,220 --> 00:26:35,720 right? 460 00:26:35,720 --> 00:26:38,300 So you don't want your thiol-reactive probe 461 00:26:38,300 --> 00:26:44,120 to react with the reducing agent in the buffer here. 462 00:26:44,120 --> 00:26:45,530 So that needs to be removed. 463 00:26:45,530 --> 00:26:49,050 And then if you remove it, you need to ask, 464 00:26:49,050 --> 00:26:55,370 does the thiol stay reduced or is it susceptible 465 00:26:55,370 --> 00:26:57,020 to air oxidation? 466 00:26:57,020 --> 00:27:00,590 So these are just all practical considerations to keep in mind. 467 00:27:00,590 --> 00:27:03,200 If a reaction doesn't work, why doesn't it? 468 00:27:03,200 --> 00:27:08,210 And was it something that wasn't right with the buffers there? 469 00:27:08,210 --> 00:27:08,960 OK. 470 00:27:08,960 --> 00:27:14,120 So back to this issue of not having much control 471 00:27:14,120 --> 00:27:18,950 about timing control for reactivity of these types 472 00:27:18,950 --> 00:27:23,250 of groups, what could be done to overcome that? 473 00:27:23,250 --> 00:27:28,250 So what other types of cross-linkers are out there? 474 00:27:28,250 --> 00:27:29,269 Yeah. 475 00:27:29,269 --> 00:27:29,810 Photo-active. 476 00:27:29,810 --> 00:27:32,460 Photo-reactive cross-linkers. 477 00:27:32,460 --> 00:27:34,310 So what's the idea here? 478 00:27:40,202 --> 00:27:47,164 AUDIENCE: [INAUDIBLE] the appropriate [INAUDIBLE] 479 00:27:47,164 --> 00:27:48,080 ELIZABETH NOLAN: Yeah. 480 00:27:48,080 --> 00:27:50,430 So what do we have? 481 00:27:50,430 --> 00:27:52,360 And what can we do? 482 00:27:52,360 --> 00:27:55,390 So just the first point to make is 483 00:27:55,390 --> 00:27:59,890 that we want to think about specific labeling here. 484 00:27:59,890 --> 00:28:19,540 So we can attach site-specifically to a protein 485 00:28:19,540 --> 00:28:26,670 or some other biomolecule, maybe it's bi-cysteine modification 486 00:28:26,670 --> 00:28:28,140 with something like a maleimide. 487 00:28:28,140 --> 00:28:32,080 Maybe it's unnatural amino acid incorporation. 488 00:28:32,080 --> 00:28:43,310 And it's chemically inert locally until irradiated. 489 00:28:46,490 --> 00:28:47,650 OK? 490 00:28:47,650 --> 00:28:53,240 And so, basically irradiating this photo-reactive 491 00:28:53,240 --> 00:28:57,110 cross-linker will activate the photo-reactive group, 492 00:28:57,110 --> 00:28:58,830 and then you get s-linking. 493 00:28:58,830 --> 00:28:59,330 OK. 494 00:28:59,330 --> 00:29:01,550 So this type of approach is often 495 00:29:01,550 --> 00:29:03,710 used to capture binding partners. 496 00:29:03,710 --> 00:29:07,130 It can be used in the test tube or in cells. 497 00:29:07,130 --> 00:29:10,100 What are the types of photo-reactive cross-linkers? 498 00:29:20,369 --> 00:29:21,414 AUDIENCE: Aryl azides. 499 00:29:21,414 --> 00:29:22,330 ELIZABETH NOLAN: Yeah. 500 00:29:22,330 --> 00:29:24,280 So aryl azides are one type. 501 00:29:28,190 --> 00:29:29,780 What's one we saw in class? 502 00:29:29,780 --> 00:29:32,000 Although we, didn't talk about photochemistry. 503 00:29:32,000 --> 00:29:33,150 Yeah, benzophenone. 504 00:29:39,890 --> 00:29:41,270 And there's some other examples. 505 00:29:41,270 --> 00:29:44,656 So what's another example? 506 00:29:44,656 --> 00:29:46,031 AUDIENCE: Fluorinated [INAUDIBLE] 507 00:29:46,031 --> 00:29:46,947 ELIZABETH NOLAN: Yeah. 508 00:29:46,947 --> 00:29:48,560 So they fall in here, right? 509 00:29:48,560 --> 00:29:50,600 So we can think about, either just 510 00:29:50,600 --> 00:29:56,330 phenyl azides or fluorinated phenyl azides. 511 00:29:56,330 --> 00:29:59,520 So another way to do this is to generate 512 00:29:59,520 --> 00:30:02,810 carbenes via diazirines here. 513 00:30:09,230 --> 00:30:13,530 We'll pretty much focus on these types, which are major types. 514 00:30:13,530 --> 00:30:15,720 So where did this idea come from? 515 00:30:19,150 --> 00:30:28,980 How new is this type of work to stick a photo-reactive group 516 00:30:28,980 --> 00:30:33,690 on a protein, and then use it in a cross-linking application? 517 00:30:33,690 --> 00:30:38,670 And where did the idea come from in the first place? 518 00:30:48,180 --> 00:30:52,800 What types of chemists often study photochemistry? 519 00:30:52,800 --> 00:30:55,450 AUDIENCE: DNA [INAUDIBLE] 520 00:30:55,450 --> 00:30:56,700 ELIZABETH NOLAN: More broadly. 521 00:31:01,000 --> 00:31:02,830 So physical organic chemistry, right? 522 00:31:02,830 --> 00:31:05,100 There's a whole component of photochemistry there. 523 00:31:08,200 --> 00:31:09,440 Let's take a vote. 524 00:31:09,440 --> 00:31:11,290 2000? 525 00:31:11,290 --> 00:31:14,480 First photo cross-linker. 526 00:31:14,480 --> 00:31:17,980 1990? 527 00:31:17,980 --> 00:31:20,384 '80? 528 00:31:20,384 --> 00:31:22,644 '70? 529 00:31:22,644 --> 00:31:24,696 '60? 530 00:31:24,696 --> 00:31:25,740 Just no clue? 531 00:31:32,320 --> 00:31:35,110 So around 1962 was the first paper 532 00:31:35,110 --> 00:31:41,030 using a photoreactive group on a protein here, Westheimer. 533 00:31:41,030 --> 00:31:44,320 And then Jeremy Nulls in 1969 was the first example 534 00:31:44,320 --> 00:31:45,260 of an aryl azide. 535 00:31:45,260 --> 00:31:45,760 OK? 536 00:31:45,760 --> 00:31:49,060 So this work came out of physical organic chemistry 537 00:31:49,060 --> 00:31:51,790 and at a time where physical organic chemists were 538 00:31:51,790 --> 00:31:55,210 transitioning into enzymology. 539 00:31:55,210 --> 00:31:58,750 So we don't have time to go into a lot of the photochemistry 540 00:31:58,750 --> 00:32:05,170 of these different moieties, but it was quite rich there. 541 00:32:05,170 --> 00:32:08,095 So how does this work? 542 00:32:12,010 --> 00:32:14,890 What types of reactions and groups 543 00:32:14,890 --> 00:32:17,930 get modified here in the cross-linking? 544 00:32:17,930 --> 00:32:19,390 So let's think about them. 545 00:32:32,070 --> 00:32:33,870 So let's consider an aryl azide. 546 00:32:42,360 --> 00:32:48,210 So what happens when aryl azides are irradiated with UV light? 547 00:32:55,725 --> 00:32:57,349 AUDIENCE: Took all of the nitrogen gas. 548 00:32:57,349 --> 00:32:59,770 Get a nitrene. 549 00:32:59,770 --> 00:33:01,681 ELIZABETH NOLAN: Get a nitrene. 550 00:33:01,681 --> 00:33:02,180 Yeah. 551 00:33:02,180 --> 00:33:04,550 So if we just think about nitrenes for a minute, 552 00:33:04,550 --> 00:33:06,560 what types of chemistry do nitrenes do? 553 00:33:17,145 --> 00:33:17,895 Are they reactive? 554 00:33:38,530 --> 00:33:42,640 So can they insert into C-H bonds? 555 00:33:42,640 --> 00:33:44,100 N-H bonds? 556 00:33:44,100 --> 00:33:45,440 Add to double bonds? 557 00:33:45,440 --> 00:33:47,000 Can they do other things as well? 558 00:33:54,780 --> 00:33:55,280 OK. 559 00:33:55,280 --> 00:33:56,810 So here we have our protein. 560 00:33:56,810 --> 00:33:57,840 What's going to happen? 561 00:33:57,840 --> 00:34:01,010 As Steve said, we're going to generate a nitrene. 562 00:34:01,010 --> 00:34:04,145 So how does that happen? 563 00:34:04,145 --> 00:34:52,090 We irradiate with light to get our nitrene. 564 00:34:52,090 --> 00:34:55,960 So what happens with these aryl azides 565 00:34:55,960 --> 00:34:58,610 is some interesting photochemistry when you're 566 00:34:58,610 --> 00:35:03,080 at, say, room temperature. 567 00:35:03,080 --> 00:35:06,710 So rather than this nitrene reacting, say, 568 00:35:06,710 --> 00:35:10,010 with a C-H bond or an N-H bond, it actually 569 00:35:10,010 --> 00:35:11,540 undergoes a ring expansion. 570 00:35:14,970 --> 00:35:18,590 So what we get-- and this is very fast. 571 00:35:22,205 --> 00:35:27,620 So on the order of 10 to 100 picoseconds. 572 00:35:27,620 --> 00:35:29,810 So this happens before it has a chance 573 00:35:29,810 --> 00:35:31,280 to react with something else. 574 00:35:50,920 --> 00:35:51,430 OK. 575 00:35:51,430 --> 00:35:53,366 To give us this intermediate. 576 00:36:00,170 --> 00:36:00,730 OK. 577 00:36:00,730 --> 00:36:03,565 And so, this species has very different chemistry 578 00:36:03,565 --> 00:36:04,190 than a nitrene. 579 00:36:06,750 --> 00:36:11,810 And what happens is it will react with nucleophiles. 580 00:36:11,810 --> 00:36:19,670 So imagine our amino group to give the cross-link. 581 00:36:48,320 --> 00:36:51,340 So this pathway is the dominant pathway 582 00:36:51,340 --> 00:36:56,220 if just an aryl azide is used here. 583 00:36:56,220 --> 00:36:57,900 To think about from the standpoint 584 00:36:57,900 --> 00:36:59,400 of wanting to do cross-linking. 585 00:36:59,400 --> 00:37:02,340 So let's say you attach this aryl azide 586 00:37:02,340 --> 00:37:07,710 to a protein of interest, and then you 587 00:37:07,710 --> 00:37:09,210 irradiate with light and look for it 588 00:37:09,210 --> 00:37:12,450 to cross-link with something, is this an issue? 589 00:37:17,050 --> 00:37:18,650 It will form a cross-link. 590 00:37:27,580 --> 00:37:31,940 Would you rather have a nitrene reactor or this seven member 591 00:37:31,940 --> 00:37:33,440 reaction with a seven membered ring? 592 00:37:36,905 --> 00:37:40,429 AUDIENCE: [INAUDIBLE] nitrene, but it 593 00:37:40,429 --> 00:37:42,845 would depend on what you're actually looking at, like what 594 00:37:42,845 --> 00:37:44,037 you were investigating. 595 00:37:44,037 --> 00:37:44,870 ELIZABETH NOLAN: OK. 596 00:37:44,870 --> 00:37:49,283 So why would you argue for the nitrene? 597 00:37:49,283 --> 00:37:52,660 AUDIENCE: Because we were talking about the nitrene 598 00:37:52,660 --> 00:38:02,295 does have the capacity to do a [INAUDIBLE] So 599 00:38:02,295 --> 00:38:05,350 if you wanted to do something like that kind of chemistry, 600 00:38:05,350 --> 00:38:11,734 then having this be the dominant pathway would be inefficient. 601 00:38:11,734 --> 00:38:12,650 ELIZABETH NOLAN: Yeah. 602 00:38:12,650 --> 00:38:15,300 There's a lot more C-H bonds than there 603 00:38:15,300 --> 00:38:18,360 are lysines or N-termini. 604 00:38:18,360 --> 00:38:20,250 So that's one aspect. 605 00:38:20,250 --> 00:38:22,020 We've lost that chemistry. 606 00:38:22,020 --> 00:38:28,950 And then to another point, how well did these reactions work? 607 00:38:28,950 --> 00:38:30,930 So nitrene reactions are very fast. 608 00:38:30,930 --> 00:38:36,270 Relatively speaking, this is kind of sluggish there. 609 00:38:36,270 --> 00:38:41,910 And so the question is, what can be done in order 610 00:38:41,910 --> 00:38:44,260 to improve upon this? 611 00:38:44,260 --> 00:38:44,760 OK. 612 00:38:44,760 --> 00:38:49,230 And Steve mentioned these fluorinated phenyl azides 613 00:38:49,230 --> 00:38:49,990 there. 614 00:38:49,990 --> 00:38:54,750 And so, photochemical work, unrelated to any sort 615 00:38:54,750 --> 00:38:58,170 of biological cross-linking chemistry, 616 00:38:58,170 --> 00:39:02,760 showed that if you fluorinate aryl azides 617 00:39:02,760 --> 00:39:07,680 you can get nitrene reactivity, rather than this other pathway 618 00:39:07,680 --> 00:39:08,231 here. 619 00:39:08,231 --> 00:39:08,730 OK? 620 00:39:08,730 --> 00:39:11,400 And so, if we just take a look at that, what happens? 621 00:39:41,890 --> 00:39:46,330 For instance, imagine we have this tetrafluoro analog here. 622 00:39:49,590 --> 00:39:54,229 We can imagine irradiating this and getting to our nitrene. 623 00:39:54,229 --> 00:39:55,395 I'm going to skip the steps. 624 00:40:17,420 --> 00:40:18,960 OK. 625 00:40:18,960 --> 00:40:19,980 Now what can happen? 626 00:40:19,980 --> 00:40:27,955 Imagine we have some C-H bond nearby. 627 00:40:44,200 --> 00:40:45,886 We get this cross-link. 628 00:40:51,600 --> 00:40:54,680 And this reaction is very, very fast here. 629 00:40:54,680 --> 00:40:55,500 Very, very fast. 630 00:41:05,150 --> 00:41:08,390 Can ring expansion occur in this situation? 631 00:41:12,061 --> 00:41:12,560 OK. 632 00:41:12,560 --> 00:41:15,140 So I'm pointing this out, because the language 633 00:41:15,140 --> 00:41:18,230 in the packet was a little strong. 634 00:41:18,230 --> 00:41:22,040 If there is something for this nitrene to react with nearby, 635 00:41:22,040 --> 00:41:23,780 it will react. 636 00:41:23,780 --> 00:41:25,880 But this can undergo ring expansion. 637 00:41:25,880 --> 00:41:28,660 It's just much slower than the case above. 638 00:41:28,660 --> 00:41:33,500 So the studies I've read say about 170-fold slower there. 639 00:41:33,500 --> 00:41:36,550 So it's not that the pathway is completely blocked. 640 00:41:36,550 --> 00:41:39,680 It also too depends on the experimental conditions. 641 00:41:39,680 --> 00:41:45,690 But anyhow, this is quoted to be near diffusion controlled here 642 00:41:45,690 --> 00:41:46,400 for that. 643 00:41:52,436 --> 00:41:54,060 I mean, this is pretty interesting when 644 00:41:54,060 --> 00:41:55,143 you think about it, right? 645 00:41:55,143 --> 00:41:59,700 Because aryl azides, they can be fed to cells 646 00:41:59,700 --> 00:42:03,580 to do unnatural amino acid incorporation, right? 647 00:42:03,580 --> 00:42:05,640 They're used in click chemistry for instance, 648 00:42:05,640 --> 00:42:07,620 types of conjugation chemistry. 649 00:42:07,620 --> 00:42:10,980 But here, the photochemistry can be taken advantage of 650 00:42:10,980 --> 00:42:14,820 to give a cross-linker that can be controlled 651 00:42:14,820 --> 00:42:18,790 in a temporal manner there. 652 00:42:18,790 --> 00:42:21,240 So what about the benzophenone? 653 00:42:21,240 --> 00:42:30,090 What does benzophenone react with after being 654 00:42:30,090 --> 00:42:32,880 irradiated with light? 655 00:42:32,880 --> 00:42:37,880 So imagine you have your protein. 656 00:42:37,880 --> 00:42:44,280 And maybe in this case you did unnatural amino acid 657 00:42:44,280 --> 00:42:48,000 incorporation to site-specifically attach 658 00:42:48,000 --> 00:42:49,110 a benzophenone. 659 00:42:52,900 --> 00:42:53,540 What happens? 660 00:43:05,370 --> 00:43:08,100 What happens is that there's formation 661 00:43:08,100 --> 00:43:09,510 of a triplet diradical. 662 00:43:25,254 --> 00:43:26,770 And what will this do? 663 00:43:29,580 --> 00:43:55,670 Here, it's going to react with some C-H bond 664 00:43:55,670 --> 00:43:56,810 to get the cross-link. 665 00:44:09,310 --> 00:44:17,920 Let's say you have this guy here and you 666 00:44:17,920 --> 00:44:21,370 want to do a cross-linking experiment. 667 00:44:21,370 --> 00:44:24,700 So we can imagine some different possibilities. 668 00:44:31,520 --> 00:44:33,030 What do you think you'll get out? 669 00:44:33,030 --> 00:44:35,940 Are you only going to get out your desired cross-link? 670 00:44:39,892 --> 00:44:41,374 What might happen? 671 00:44:46,810 --> 00:44:50,155 2:00, 1:50 on a Friday. 672 00:44:50,155 --> 00:44:52,180 Let's get some jumping jacks. 673 00:44:52,180 --> 00:44:52,810 Come on. 674 00:45:00,474 --> 00:45:04,810 Should I dismiss all of you, because there is a major energy 675 00:45:04,810 --> 00:45:08,910 low today, I have to say there. 676 00:45:08,910 --> 00:45:09,756 Yeah. 677 00:45:09,756 --> 00:45:14,378 Do you think you'll get one product, 10 products, 100? 678 00:45:14,378 --> 00:45:16,450 AUDIENCE: There will be lots of side reactions. 679 00:45:16,450 --> 00:45:17,408 ELIZABETH NOLAN: Right. 680 00:45:17,408 --> 00:45:20,020 There's still the possibility for many side reactions, right? 681 00:45:20,020 --> 00:45:21,686 And you always need to be aware of that. 682 00:45:21,686 --> 00:45:24,590 So if you cross-link something, the next question is, 683 00:45:24,590 --> 00:45:27,250 is this something that's actually relevant or not? 684 00:45:27,250 --> 00:45:30,050 Or is it an artifact there? 685 00:45:30,050 --> 00:45:34,606 So the analysis can be very complicated. 686 00:45:34,606 --> 00:45:36,480 And so, that's just something to think about. 687 00:45:36,480 --> 00:45:39,850 Say you have cross-linked species from cell lysate, 688 00:45:39,850 --> 00:45:43,380 what are you going to do to analyze that? 689 00:45:54,120 --> 00:45:56,460 Just think about some of the things that have come up 690 00:45:56,460 --> 00:45:58,000 in other contexts here. 691 00:45:58,000 --> 00:46:01,320 We talked about protease digest and mass spec 692 00:46:01,320 --> 00:46:04,030 for looking at substrates of GroEL GroES, that's 693 00:46:04,030 --> 00:46:05,280 something that can be applied. 694 00:46:05,280 --> 00:46:09,330 And there's many sophisticated new tools 695 00:46:09,330 --> 00:46:13,200 to get a lot of information out of the mass spec, which 696 00:46:13,200 --> 00:46:14,460 we won't talk about. 697 00:46:14,460 --> 00:46:19,530 But having tags within the cross-linker, right? 698 00:46:19,530 --> 00:46:22,560 So then you need to ask, how well is the coverage going 699 00:46:22,560 --> 00:46:23,550 to be? 700 00:46:23,550 --> 00:46:28,180 So even after this step, there's a lot more work, 701 00:46:28,180 --> 00:46:33,300 which we won't go into details in this recitation today. 702 00:46:36,590 --> 00:46:40,830 What about inherent efficiency of cross-linking 703 00:46:40,830 --> 00:46:44,870 in terms of these benzophenone versus the aryl azides? 704 00:46:47,510 --> 00:46:51,420 We want to think about relative cross-linking efficiency. 705 00:46:51,420 --> 00:46:52,530 Any sense of that? 706 00:46:57,289 --> 00:46:59,080 AUDIENCE: I think the benzophenone compared 707 00:46:59,080 --> 00:47:01,414 to the diazirine is a lot less efficient. 708 00:47:01,414 --> 00:47:03,794 I don't really know [INAUDIBLE] 709 00:47:03,794 --> 00:47:05,590 AUDIENCE: I have a question. 710 00:47:05,590 --> 00:47:07,120 When we're talking about efficiency, 711 00:47:07,120 --> 00:47:09,460 is it purely based on the speed of this reactivity? 712 00:47:09,460 --> 00:47:10,970 Or is it also taking into account 713 00:47:10,970 --> 00:47:14,008 the different cross-reactions that could occur? 714 00:47:14,008 --> 00:47:16,735 Because it seems like there are more possibilities 715 00:47:16,735 --> 00:47:17,776 for more cross-reactions. 716 00:47:17,776 --> 00:47:21,552 Even though it might be more reactive, it's not-- 717 00:47:24,270 --> 00:47:26,120 ELIZABETH NOLAN: Yeah. 718 00:47:26,120 --> 00:47:27,240 The former, right? 719 00:47:27,240 --> 00:47:29,590 Just thinking about the reaction. 720 00:47:29,590 --> 00:47:31,500 There's the possibility of cross-reactions 721 00:47:31,500 --> 00:47:32,210 for all of these. 722 00:47:32,210 --> 00:47:33,370 They're highly reactive. 723 00:47:33,370 --> 00:47:34,950 A nitrene is highly reactive. 724 00:47:34,950 --> 00:47:37,500 The benzophenone triplet diradical is highly reactive. 725 00:47:37,500 --> 00:47:41,400 A carbene, if you're going to get that from some diazirine 726 00:47:41,400 --> 00:47:43,640 is very reactive. 727 00:47:43,640 --> 00:47:45,390 And yes, it's something important to think 728 00:47:45,390 --> 00:47:47,710 about in terms of your experiment. 729 00:47:47,710 --> 00:47:50,760 What is the relative efficiency of the reaction? 730 00:47:50,760 --> 00:47:52,787 So I said that aryl azide is a little sluggish 731 00:47:52,787 --> 00:47:53,745 compared to the others. 732 00:47:57,750 --> 00:47:59,340 Something to consider, right? 733 00:47:59,340 --> 00:48:01,830 You know what is the timescale of whatever it 734 00:48:01,830 --> 00:48:04,790 is you're trying to trap. 735 00:48:04,790 --> 00:48:05,640 So the wavelengths. 736 00:48:05,640 --> 00:48:08,820 What is it about these wavelengths 737 00:48:08,820 --> 00:48:10,470 that might be undesirable? 738 00:48:15,390 --> 00:48:19,110 AUDIENCE: For in vivo studies, one shifting towards UV 739 00:48:19,110 --> 00:48:22,260 means that you can have issues undesirable, 740 00:48:22,260 --> 00:48:24,120 like DNA cross-linking stuff, but also it 741 00:48:24,120 --> 00:48:25,740 means that it's not going to have 742 00:48:25,740 --> 00:48:31,270 deep penetrants [INAUDIBLE] shift towards [INAUDIBLE].. 743 00:48:31,270 --> 00:48:33,570 ELIZABETH NOLAN: What wavelength would you like? 744 00:48:33,570 --> 00:48:36,670 JOANNE STUBBE: I would like it around 650. 745 00:48:36,670 --> 00:48:39,791 These are all UV visible interface. 746 00:48:39,791 --> 00:48:41,290 And you have hundreds of things that 747 00:48:41,290 --> 00:48:45,566 absorb length are very incredible inefficient. 748 00:48:45,566 --> 00:48:48,940 [INAUDIBLE] Most people never identify what 749 00:48:48,940 --> 00:48:50,614 they get out of the other side. 750 00:48:50,614 --> 00:48:52,984 They just see two things stuck together, 751 00:48:52,984 --> 00:48:55,828 and that's the extent of it. 752 00:48:55,828 --> 00:48:58,372 They never describe the molecular details. 753 00:48:58,372 --> 00:49:00,330 ELIZABETH NOLAN: So let's actually just close-- 754 00:49:00,330 --> 00:49:02,642 JOANNE STUBBE: [INAUDIBLE] 755 00:49:02,642 --> 00:49:03,600 ELIZABETH NOLAN: Right. 756 00:49:03,600 --> 00:49:06,570 So one of the questions I asked in the discussion section, 757 00:49:06,570 --> 00:49:10,770 is it worth the effort if you're going to site-specifically put 758 00:49:10,770 --> 00:49:11,610 in a cross-linker? 759 00:49:11,610 --> 00:49:16,380 And imagine you find this protein-protein interaction, 760 00:49:16,380 --> 00:49:18,174 if one chooses, you can do quite a bit 761 00:49:18,174 --> 00:49:19,590 more experiments in terms of where 762 00:49:19,590 --> 00:49:24,000 you place this cross-linker and mapping out that interaction 763 00:49:24,000 --> 00:49:24,840 region there. 764 00:49:24,840 --> 00:49:27,900 And so, that's I think also just a take-home is often 765 00:49:27,900 --> 00:49:31,710 you need to put your reactive group in more than one place 766 00:49:31,710 --> 00:49:35,840 to really get at the answer to the question you're asking. 767 00:49:35,840 --> 00:49:40,770 And so, there's folks around doing that there. 768 00:49:40,770 --> 00:49:42,300 But is it 20 positions? 769 00:49:42,300 --> 00:49:43,440 Is it 10? 770 00:49:43,440 --> 00:49:44,629 Is it 50? 771 00:49:44,629 --> 00:49:46,420 Because if you don't know at the beginning, 772 00:49:46,420 --> 00:49:49,950 you may need to do a lot of just systematic trial and error 773 00:49:49,950 --> 00:49:51,750 for that. 774 00:49:51,750 --> 00:49:52,530 Yeah. 775 00:49:52,530 --> 00:49:55,860 So I think you should all read the packet. 776 00:49:55,860 --> 00:50:00,720 And there are some suggestions for reading 777 00:50:00,720 --> 00:50:02,640 if you're curious to learn more, one of which 778 00:50:02,640 --> 00:50:04,230 is a manual from Thermo. 779 00:50:04,230 --> 00:50:08,280 So often, the companies give a lot of good general background 780 00:50:08,280 --> 00:50:10,860 information, and there's many different types of chemistry 781 00:50:10,860 --> 00:50:12,330 included in that as well. 782 00:50:12,330 --> 00:50:14,520 I'll also point out, Ed is here for those of you 783 00:50:14,520 --> 00:50:15,570 who don't know Ed. 784 00:50:15,570 --> 00:50:18,690 So he'll be presenting next week on cryo-EM. 785 00:50:18,690 --> 00:50:22,710 And you should definitely read the fatty acid synthase paper 786 00:50:22,710 --> 00:50:23,860 beforehand. 787 00:50:23,860 --> 00:50:25,680 The structures are incredible. 788 00:50:25,680 --> 00:50:28,500 And fatty acid synthase serves as a base 789 00:50:28,500 --> 00:50:32,020 for our discussions of polyketide and polyketide 790 00:50:32,020 --> 00:50:36,210 synthases, which is where we'll begin module four in thinking 791 00:50:36,210 --> 00:50:39,540 about the biosynthesis of natural products there. 792 00:50:39,540 --> 00:50:40,040 OK. 793 00:50:40,040 --> 00:50:42,490 Have a good weekend.