1 00:00:00,960 --> 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 --> 00:00:11,470 continue to offer high quality educational resources for free. 5 00:00:11,470 --> 00:00:14,100 To make a donation or view additional materials 6 00:00:14,100 --> 00:00:18,050 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:18,050 --> 00:00:19,260 at ocw.mit.edu. 8 00:00:24,730 --> 00:00:27,130 JOANNE STUBBE: So the key question is, do these-- 9 00:00:27,130 --> 00:00:30,970 and I think this is a general question you can ask, 10 00:00:30,970 --> 00:00:35,110 metabolically, inside any cell is 11 00:00:35,110 --> 00:00:38,980 do these enzymes that are on different polypeptides cluster. 12 00:00:38,980 --> 00:00:42,700 And is there an advantage, kinetically or whatever, 13 00:00:42,700 --> 00:00:44,590 is there some kind of an advantage 14 00:00:44,590 --> 00:00:47,710 to have clustering inside the cell. 15 00:00:47,710 --> 00:00:50,480 And where have you seen something like this before? 16 00:00:50,480 --> 00:00:51,340 Do you remember? 17 00:00:51,340 --> 00:00:53,590 Do you remember the section. 18 00:00:53,590 --> 00:00:58,270 Where have you see multi enzyme complexes and clustering 19 00:00:58,270 --> 00:01:00,900 before? 20 00:01:00,900 --> 00:01:02,250 Yeah? 21 00:01:02,250 --> 00:01:07,270 So or the classic one, PKS has been around. 22 00:01:07,270 --> 00:01:11,140 But it's completely analogous to fatty acid synthesis, right? 23 00:01:11,140 --> 00:01:14,350 And so in bacteria, they're all single polypeptides. 24 00:01:14,350 --> 00:01:17,740 In humans, they're all activities 25 00:01:17,740 --> 00:01:19,960 that are on single chains. 26 00:01:19,960 --> 00:01:22,780 OK, so that's sort of what's going on here 27 00:01:22,780 --> 00:01:24,520 with the purine pathway. 28 00:01:24,520 --> 00:01:27,820 We'll see there are ten. 29 00:01:27,820 --> 00:01:29,980 This just sort of helps us focus if we 30 00:01:29,980 --> 00:01:33,210 get to the data at the end, which I think we will 31 00:01:33,210 --> 00:01:35,310 from what we did the last time. 32 00:01:35,310 --> 00:01:43,090 Is that you have six enzymes for 10 activities. 33 00:01:43,090 --> 00:01:46,090 So that just means you have more than one enzyme per 34 00:01:46,090 --> 00:01:47,660 polypeptide, OK? 35 00:01:47,660 --> 00:01:52,780 And so I guess the key thing that I wanted to focus on 36 00:01:52,780 --> 00:01:58,110 is do you think it's important to cluster? 37 00:01:58,110 --> 00:02:00,310 Here's a pathway. 38 00:02:00,310 --> 00:02:01,163 These are the names. 39 00:02:01,163 --> 00:02:02,830 We're not going to go through the names. 40 00:02:02,830 --> 00:02:05,122 The names really aren't important for what we're doing. 41 00:02:05,122 --> 00:02:07,870 There'll be two names that we'll be looking at over and over 42 00:02:07,870 --> 00:02:08,889 again. 43 00:02:08,889 --> 00:02:12,130 These are the papers that you guys did, in fact, read. 44 00:02:12,130 --> 00:02:15,490 One is the original paper, which got a lot of press. 45 00:02:15,490 --> 00:02:18,730 And I just want to show you that there have been there's 46 00:02:18,730 --> 00:02:20,890 actually been four papers published in the last six 47 00:02:20,890 --> 00:02:22,850 months on this topic. 48 00:02:22,850 --> 00:02:25,460 And one of which was published. 49 00:02:25,460 --> 00:02:25,960 Yeah. 50 00:02:25,960 --> 00:02:29,260 One of which is published with Science, where they are now 51 00:02:29,260 --> 00:02:34,700 claiming that this complex is localized to the mitochondria. 52 00:02:34,700 --> 00:02:36,410 OK, so you take pictures. 53 00:02:36,410 --> 00:02:42,650 And it's that this is looking at super resolution fluorescence 54 00:02:42,650 --> 00:02:43,150 methods. 55 00:02:43,150 --> 00:02:48,070 And you can clearly see clumps of blobs 56 00:02:48,070 --> 00:02:49,990 focused on the mitochondria. 57 00:02:49,990 --> 00:02:52,310 Why would you want it to be at the mitochondria? 58 00:02:54,910 --> 00:02:56,530 So then you have to ask your question. 59 00:02:56,530 --> 00:02:58,180 You might need purines, because that's 60 00:02:58,180 --> 00:03:01,120 where you make, through a proto mode of force in respiration. 61 00:03:01,120 --> 00:03:04,272 Remember, when you convert oxygen to water, 62 00:03:04,272 --> 00:03:05,980 you get a huge amount of energy released. 63 00:03:05,980 --> 00:03:06,790 You make ATP. 64 00:03:06,790 --> 00:03:08,570 But it's going to be made from something. 65 00:03:08,570 --> 00:03:09,610 So maybe you would want. 66 00:03:09,610 --> 00:03:11,530 That's the way they rationalize it. 67 00:03:11,530 --> 00:03:12,400 And they do. 68 00:03:12,400 --> 00:03:16,450 And then they connect it to the other latest hot topic, which 69 00:03:16,450 --> 00:03:20,260 is EM torque, which is the major signaling switch 70 00:03:20,260 --> 00:03:23,650 for fatty acids and for amino acids. 71 00:03:23,650 --> 00:03:27,790 And now in the last two years, purines and pyrimidines, 72 00:03:27,790 --> 00:03:28,480 I decided-- 73 00:03:28,480 --> 00:03:30,855 I've done a lot of reading about it, decided and believe. 74 00:03:30,855 --> 00:03:31,750 I mean, I believe it. 75 00:03:31,750 --> 00:03:34,270 But I don't believe the connections yet. 76 00:03:34,270 --> 00:03:35,890 So again, this is what you're going 77 00:03:35,890 --> 00:03:38,890 to see in the next decade is connecting 78 00:03:38,890 --> 00:03:42,100 signaling to primary metabolic pathways, 79 00:03:42,100 --> 00:03:43,180 like the purine pathway. 80 00:03:43,180 --> 00:03:44,763 That's going to be a big thing and how 81 00:03:44,763 --> 00:03:47,050 do you connect them is going to be the key question. 82 00:03:47,050 --> 00:03:49,420 So anybody that wants to do some more reading, 83 00:03:49,420 --> 00:03:50,830 this is an updated version. 84 00:03:50,830 --> 00:03:54,260 I kept updating this three or four times. 85 00:03:54,260 --> 00:03:57,490 And so I think these are the key questions we want to focus on. 86 00:03:57,490 --> 00:03:58,930 And so what I'm going to do. 87 00:03:58,930 --> 00:04:01,920 Well, define the questions a little bit 88 00:04:01,920 --> 00:04:04,840 and whether the things we need to think about just 89 00:04:04,840 --> 00:04:09,090 to determine whether this is really important, biologically. 90 00:04:09,090 --> 00:04:12,280 Then we'll define fluorescence and what 91 00:04:12,280 --> 00:04:14,620 you can do with fluorescence. 92 00:04:14,620 --> 00:04:16,805 And then we'll come back and look at the data. 93 00:04:16,805 --> 00:04:18,430 In the paper, we're also going to look. 94 00:04:18,430 --> 00:04:20,847 We probably won't get all the way through all of the data. 95 00:04:20,847 --> 00:04:22,510 But we will look at some of that data 96 00:04:22,510 --> 00:04:26,350 again in either the next lecture or Wednesday's lecture. 97 00:04:26,350 --> 00:04:30,380 So you will see it again if we don't get through the data. 98 00:04:30,380 --> 00:04:35,050 So they claim they have a multi enzyme complex. 99 00:04:35,050 --> 00:04:38,300 Did you believe that from the data? 100 00:04:38,300 --> 00:04:40,970 I mean, they didn't look at all 10 enzymes simultaneously, 101 00:04:40,970 --> 00:04:42,850 right? 102 00:04:42,850 --> 00:04:43,505 Or six enzymes. 103 00:04:46,275 --> 00:04:48,695 AUDIENCE: Whenever they show an image of the cells, 104 00:04:48,695 --> 00:04:51,070 and then they fluorescent, trying to show local sections, 105 00:04:51,070 --> 00:04:53,983 it's always so hard for me to figure out-- 106 00:04:53,983 --> 00:04:55,150 JOANNE STUBBE: What you see. 107 00:04:55,150 --> 00:04:55,775 AUDIENCE: Yeah. 108 00:04:55,775 --> 00:04:58,420 JOANNE STUBBE: OK, so that was said. 109 00:04:58,420 --> 00:05:00,060 We'll look at some of those pictures. 110 00:05:00,060 --> 00:05:02,420 But I completely agree with that, 111 00:05:02,420 --> 00:05:04,780 that you can't see anything from fluorescence pictures. 112 00:05:04,780 --> 00:05:08,800 So everybody, all chemists or chemical biologists, 113 00:05:08,800 --> 00:05:12,580 now have huge numbers of these pictures in their papers. 114 00:05:12,580 --> 00:05:17,190 And with Alice's group, I'm always on their case 115 00:05:17,190 --> 00:05:18,850 that I can't tell a damn thing. 116 00:05:18,850 --> 00:05:19,690 This is on thesis. 117 00:05:19,690 --> 00:05:20,930 I can't see anything. 118 00:05:20,930 --> 00:05:23,800 And Alice says she can't see anything either. 119 00:05:23,800 --> 00:05:27,490 So it's very hard to see things in these pictures. 120 00:05:27,490 --> 00:05:28,870 The contrast isn't very good. 121 00:05:28,870 --> 00:05:32,620 And what her lab now does is it goes to EM, where you can 122 00:05:32,620 --> 00:05:34,420 see things much more clearly. 123 00:05:34,420 --> 00:05:35,890 The fluorescence things are tough. 124 00:05:35,890 --> 00:05:38,220 So you're not the only one. 125 00:05:38,220 --> 00:05:40,655 And if somebody says it's obvious that this. 126 00:05:40,655 --> 00:05:41,530 And you don't see it. 127 00:05:41,530 --> 00:05:43,197 Raise your hand and say, I don't see it. 128 00:05:43,197 --> 00:05:45,160 Show me what I should be looking at, OK? 129 00:05:45,160 --> 00:05:47,830 So that's a good take home message, 130 00:05:47,830 --> 00:05:50,890 because everybody and his brother is doing this. 131 00:05:50,890 --> 00:05:53,430 And this goes back to knowing how to do it correctly. 132 00:05:53,430 --> 00:05:55,430 We're not going to talk about any of that stuff. 133 00:05:55,430 --> 00:05:58,000 I mean every one of the methods I'll sort of show you 134 00:05:58,000 --> 00:05:58,910 that's out there. 135 00:05:58,910 --> 00:06:02,860 You have to really study it to make sure you're handling it 136 00:06:02,860 --> 00:06:03,490 correctly. 137 00:06:03,490 --> 00:06:07,622 So I mean I think, to me, this has been a problem 138 00:06:07,622 --> 00:06:08,830 that I've been interested in. 139 00:06:08,830 --> 00:06:11,050 And I started working on this a long time ago 140 00:06:11,050 --> 00:06:15,700 in the purine pathway is not are things 141 00:06:15,700 --> 00:06:17,212 sticking together important. 142 00:06:17,212 --> 00:06:19,045 Actually, I don't think those are important. 143 00:06:19,045 --> 00:06:22,120 You immunoprecipitate all these things. 144 00:06:22,120 --> 00:06:25,340 OK, so you say obviously these are talking to each other. 145 00:06:25,340 --> 00:06:27,760 But the key thing is the kinetic competence. 146 00:06:27,760 --> 00:06:30,980 And lots of times when you mess around. 147 00:06:30,980 --> 00:06:31,930 You get it in a state. 148 00:06:31,930 --> 00:06:34,120 And you post-translationally modify it. 149 00:06:34,120 --> 00:06:36,370 So it's sitting in this state there probably 150 00:06:36,370 --> 00:06:38,650 isn't on the pathway. 151 00:06:38,650 --> 00:06:41,920 You need to then show it's on the pathway. 152 00:06:41,920 --> 00:06:45,050 So I think a lot of protein, protein interactions, 153 00:06:45,050 --> 00:06:47,500 especially now that we know that proteins move around. 154 00:06:47,500 --> 00:06:48,790 And they're in this complex. 155 00:06:48,790 --> 00:06:49,990 And they're in that complex. 156 00:06:49,990 --> 00:06:51,350 And they're in that complex. 157 00:06:51,350 --> 00:06:53,700 The key, I think, is to transient interactions. 158 00:06:53,700 --> 00:06:55,580 So why? 159 00:06:55,580 --> 00:06:57,340 So this is just my personal take on this. 160 00:06:57,340 --> 00:06:59,320 I'm letting you think about this. 161 00:06:59,320 --> 00:07:03,260 But is it easy to look at transient interactions? 162 00:07:03,260 --> 00:07:04,420 No. 163 00:07:04,420 --> 00:07:06,750 OK, so anyhow, I think people need 164 00:07:06,750 --> 00:07:10,290 to start doing a lot more thinking about how 165 00:07:10,290 --> 00:07:11,040 to look at that. 166 00:07:11,040 --> 00:07:13,740 And one way you could look at transient interactions 167 00:07:13,740 --> 00:07:16,530 is if you can fluorescent label something. 168 00:07:16,530 --> 00:07:19,050 And they come together on a certain timescale 169 00:07:19,050 --> 00:07:20,810 and then move apart. 170 00:07:20,810 --> 00:07:23,010 And can you do that inside the cell 171 00:07:23,010 --> 00:07:27,630 with the right spatial and time resolution. 172 00:07:27,630 --> 00:07:29,820 You might be able to start looking at that. 173 00:07:29,820 --> 00:07:32,220 So that the methods that are being developed 174 00:07:32,220 --> 00:07:35,170 and continue to be developed are incredibly powerful. 175 00:07:35,170 --> 00:07:40,980 And I think will allow us to ask this question happens 176 00:07:40,980 --> 00:07:43,050 inside the cell, which you've all seen pictures 177 00:07:43,050 --> 00:07:45,090 in your introductory courses of, man, 178 00:07:45,090 --> 00:07:47,690 how complicated the inside of the cell is. 179 00:07:47,690 --> 00:07:49,720 That's part of the issue. 180 00:07:49,720 --> 00:07:55,110 So the issue is that you might have a purinosome somewhere 181 00:07:55,110 --> 00:07:57,450 in the cell, depending on the growth conditions. 182 00:07:57,450 --> 00:08:00,310 But those enzymes might be involved in other things. 183 00:08:00,310 --> 00:08:02,370 And so you have only a tiny amount of it, 184 00:08:02,370 --> 00:08:06,420 as opposed to trying to make the cell by growth conditions, 185 00:08:06,420 --> 00:08:08,350 putting it into all one state. 186 00:08:08,350 --> 00:08:09,460 So you can see it. 187 00:08:09,460 --> 00:08:12,360 So the question is, how do you see it? 188 00:08:12,360 --> 00:08:14,680 And so that's the key issue. 189 00:08:14,680 --> 00:08:16,290 And if you perturb it enough. 190 00:08:16,290 --> 00:08:17,618 And you do see it. 191 00:08:17,618 --> 00:08:19,035 Then you have to ask the question. 192 00:08:19,035 --> 00:08:21,930 And this is a question that you might 193 00:08:21,930 --> 00:08:23,670 want to think about in terms of these two 194 00:08:23,670 --> 00:08:25,770 papers you were reading. 195 00:08:25,770 --> 00:08:27,390 That's what. 196 00:08:27,390 --> 00:08:29,640 If you do this, that's what the Marcotte paper 197 00:08:29,640 --> 00:08:33,120 said, that the cells were incredibly sick when 198 00:08:33,120 --> 00:08:35,190 you take out all the purines. 199 00:08:35,190 --> 00:08:39,570 And in fact, Alice is-- because of this mitochondria connection 200 00:08:39,570 --> 00:08:43,049 between the purinosome and Alice's interest 201 00:08:43,049 --> 00:08:47,580 in the mitochondria, she's had people trying to repeat this. 202 00:08:47,580 --> 00:08:50,982 And Vicki Hung worked on this and couldn't repeat it. 203 00:08:50,982 --> 00:08:52,690 So she didn't spend that much time on it. 204 00:08:52,690 --> 00:08:55,830 But all I'm saying is it's not a slam dunk 205 00:08:55,830 --> 00:08:56,880 to be able to do this. 206 00:08:56,880 --> 00:08:58,980 But that being said, I think this 207 00:08:58,980 --> 00:09:00,660 has been an issue that people have been 208 00:09:00,660 --> 00:09:02,100 thinking about for decades. 209 00:09:02,100 --> 00:09:05,280 And it's just really hard to test experimentally 210 00:09:05,280 --> 00:09:06,730 inside the cell. 211 00:09:06,730 --> 00:09:09,390 This is where we need chemical biologists to figure out 212 00:09:09,390 --> 00:09:11,310 new ways of being able to look at this, 213 00:09:11,310 --> 00:09:17,110 so that you can actually make a measurement that's interesting. 214 00:09:17,110 --> 00:09:20,400 So I guess the question I want to start 215 00:09:20,400 --> 00:09:22,770 with, before we researched looking at fluorescence, 216 00:09:22,770 --> 00:09:25,600 is why do you think it would be important to do this. 217 00:09:25,600 --> 00:09:29,590 Or do you think it would be important to have a complex. 218 00:09:29,590 --> 00:09:31,920 What's the advantage of doing that? 219 00:09:31,920 --> 00:09:32,970 Yeah. 220 00:09:32,970 --> 00:09:34,470 AUDIENCE: You were saying in lecture 221 00:09:34,470 --> 00:09:36,772 that you want to increase the effective molarity. 222 00:09:36,772 --> 00:09:40,050 And so by having all these things right next to each 223 00:09:40,050 --> 00:09:44,930 other, there's-- obviously you're going to have more 224 00:09:44,930 --> 00:09:47,787 interactions per second. 225 00:09:47,787 --> 00:09:49,870 JOANNE STUBBE: Well, you may or may-- you may not. 226 00:09:49,870 --> 00:09:52,020 It depends. 227 00:09:52,020 --> 00:09:54,690 So I think this is the key question. 228 00:09:54,690 --> 00:09:59,805 Is diffusion fast inside the cell? 229 00:09:59,805 --> 00:10:00,800 AUDIENCE: Yeah. 230 00:10:00,800 --> 00:10:00,886 JOANNE STUBBE: Yeah. 231 00:10:00,886 --> 00:10:02,010 It's still very fast. 232 00:10:02,010 --> 00:10:04,070 For small molecules, it's incredibly fast. 233 00:10:04,070 --> 00:10:06,160 Even for proteins, it's incredibly fast. 234 00:10:06,160 --> 00:10:08,340 So even if this guy is over here. 235 00:10:08,340 --> 00:10:10,853 If you're turning over here at a much slower rate, 236 00:10:10,853 --> 00:10:12,270 and you have enough of them so you 237 00:10:12,270 --> 00:10:14,370 can interact at diffusion control, 238 00:10:14,370 --> 00:10:16,770 do you need this organization? 239 00:10:16,770 --> 00:10:20,010 There are a lot of smart people who think you don't need that. 240 00:10:20,010 --> 00:10:23,130 There are a lot of smart people who think you do need that. 241 00:10:23,130 --> 00:10:25,470 But this is the question I want to raise. 242 00:10:25,470 --> 00:10:29,760 However, so catalytic efficiency is absolutely it. 243 00:10:29,760 --> 00:10:33,600 But where might you really need catalytic efficiency. 244 00:10:33,600 --> 00:10:35,280 And so that goes back. 245 00:10:35,280 --> 00:10:37,350 There are places where you really need this. 246 00:10:39,880 --> 00:10:41,895 So if you look at the first intermediate 247 00:10:41,895 --> 00:10:47,630 in the pathway, this guy, what do you think about that guy? 248 00:10:47,630 --> 00:10:49,183 Do you think he's stable? 249 00:10:49,183 --> 00:10:50,850 So if you look at the first intermediate 250 00:10:50,850 --> 00:10:53,660 in the pathway, which we'll talk about next time. 251 00:10:53,660 --> 00:10:59,885 So this is amino phosphoribosine-- 252 00:10:59,885 --> 00:11:01,990 phospho-- I'm drawing a complete-- 253 00:11:01,990 --> 00:11:04,090 I think I'm tired. 254 00:11:04,090 --> 00:11:08,290 Anyhow it's the amino analogue of ribose 5-phosphate. 255 00:11:08,290 --> 00:11:11,770 Phosphoribosylamine, that's what it's called, PRA. 256 00:11:11,770 --> 00:11:15,530 OK, do you think that's stable, as chemists? 257 00:11:15,530 --> 00:11:18,822 So what do you think that could do? 258 00:11:18,822 --> 00:11:22,139 AUDIENCE: Could you release the amine? 259 00:11:22,139 --> 00:11:24,056 JOANNE STUBBE: Yeah, so how would you do that? 260 00:11:24,056 --> 00:11:25,431 AUDIENCE: So if it's proteinated, 261 00:11:25,431 --> 00:11:27,027 and then the ring opens till-- 262 00:11:27,027 --> 00:11:28,860 JOANNE STUBBE: OK, so that would be one way. 263 00:11:28,860 --> 00:11:30,680 You want to release it that way. 264 00:11:30,680 --> 00:11:34,050 OK, so it would have to be under conditions. 265 00:11:34,050 --> 00:11:36,060 We could do that under neutral conditions. 266 00:11:36,060 --> 00:11:37,490 What else can happen to this ring. 267 00:11:40,600 --> 00:11:43,423 That doesn't happen. 268 00:11:43,423 --> 00:11:45,590 There are lots of ways this molecule can break down. 269 00:11:45,590 --> 00:11:49,930 OK, it depends on the details of the environment. 270 00:11:49,930 --> 00:11:52,030 How else could this molecule ring open? 271 00:11:56,110 --> 00:11:57,810 You wouldn't need to ring open here. 272 00:11:57,810 --> 00:12:01,800 You just go through an oxocarbenium ion 273 00:12:01,800 --> 00:12:03,180 and have water attack. 274 00:12:03,180 --> 00:12:05,898 So what if it opens that way. 275 00:12:05,898 --> 00:12:07,440 So that's the way you form aldehydes. 276 00:12:07,440 --> 00:12:10,650 All sugars are in equilibrium with aldehydes. 277 00:12:10,650 --> 00:12:12,040 These things are in equilibrium. 278 00:12:12,040 --> 00:12:13,740 So you have a ring open species. 279 00:12:13,740 --> 00:12:16,800 But then what happens if a ring closes? 280 00:12:16,800 --> 00:12:20,780 It can ring close from the top face or the bottom face. 281 00:12:20,780 --> 00:12:21,590 You have an imine. 282 00:12:21,590 --> 00:12:23,305 What can happen to the imine? 283 00:12:23,305 --> 00:12:25,130 It can hydrolyze. 284 00:12:25,130 --> 00:12:28,190 This molecule, and this is a molecule 285 00:12:28,190 --> 00:12:31,040 my lab worked on decades ago, has a half life 286 00:12:31,040 --> 00:12:34,260 in solution of 10 seconds. 287 00:12:34,260 --> 00:12:37,070 So is 10 seconds short or long, biologically? 288 00:12:42,770 --> 00:12:43,717 What do you think? 289 00:12:43,717 --> 00:12:44,194 AUDIENCE: I'm going to say short. 290 00:12:44,194 --> 00:12:45,150 But I don't know 291 00:12:45,150 --> 00:12:45,983 JOANNE STUBBE: Yeah. 292 00:12:45,983 --> 00:12:48,230 I think it's amazingly long inside the cell. 293 00:12:48,230 --> 00:12:50,763 So I think as a chemist, nobody could ever. 294 00:12:50,763 --> 00:12:52,180 Nobody ever saw this intermediate. 295 00:12:52,180 --> 00:12:55,033 My lab was the first one that figured out how to look at it. 296 00:12:55,033 --> 00:12:56,200 And I won't go through that. 297 00:12:56,200 --> 00:13:01,510 But the fact is that 10 seconds is a long time inside the cell, 298 00:13:01,510 --> 00:13:03,250 if you think about how small the cell is 299 00:13:03,250 --> 00:13:05,520 and how fast diffusion is. 300 00:13:05,520 --> 00:13:07,510 OK, so one place, though, where you 301 00:13:07,510 --> 00:13:11,890 might want to have organization is if you have something 302 00:13:11,890 --> 00:13:14,120 chemically really unstable. 303 00:13:14,120 --> 00:13:16,710 OK, because then when you generate it, 304 00:13:16,710 --> 00:13:18,630 it could potentially be passed off, 305 00:13:18,630 --> 00:13:22,510 or as you say in the immediate vicinity, it's a competition. 306 00:13:22,510 --> 00:13:25,150 But if it's right there, you're effective molarity, 307 00:13:25,150 --> 00:13:27,030 that would get into that first question, 308 00:13:27,030 --> 00:13:28,330 the effective molarity. 309 00:13:28,330 --> 00:13:32,100 It would be high enough to get passed on. 310 00:13:32,100 --> 00:13:33,730 It would get high enough to get passed 311 00:13:33,730 --> 00:13:38,200 on to the next guy in the pathway. 312 00:13:38,200 --> 00:13:42,760 So that would be one thing is instability. 313 00:13:42,760 --> 00:13:44,320 And in the purine pathway. 314 00:13:44,320 --> 00:13:46,150 We will go through this a little bit. 315 00:13:46,150 --> 00:13:48,010 But really, that's one of the things that's 316 00:13:48,010 --> 00:13:51,270 most amazing about Buchanan's elucidations of the pathway 317 00:13:51,270 --> 00:13:53,570 is only intermediates are unstable. 318 00:13:53,570 --> 00:13:56,350 Nobody, still, if you're looking at omics, 319 00:13:56,350 --> 00:13:58,522 looking at nucleotides, nobody knows how 320 00:13:58,522 --> 00:13:59,730 to deal with these molecules. 321 00:13:59,730 --> 00:14:01,900 They're all chemically unstable. 322 00:14:01,900 --> 00:14:04,308 And they don't get that they're chemically unstable. 323 00:14:04,308 --> 00:14:05,350 They don't ever see them. 324 00:14:05,350 --> 00:14:06,220 The reason they don't see them is 325 00:14:06,220 --> 00:14:07,762 because they don't know how to handle 326 00:14:07,762 --> 00:14:11,340 them to keep them alive during the analysis 327 00:14:11,340 --> 00:14:12,650 part of the project 328 00:14:12,650 --> 00:14:15,540 OK, so you have this instability problem. 329 00:14:15,540 --> 00:14:19,110 And in the purine pathway, the instability problem 330 00:14:19,110 --> 00:14:21,610 is a real problem for not just this guy. 331 00:14:21,610 --> 00:14:22,840 This guy is obvious. 332 00:14:22,840 --> 00:14:24,170 But for other guys. 333 00:14:24,170 --> 00:14:28,090 OK, so then the next question is where else. 334 00:14:28,090 --> 00:14:31,840 And if you're thinking about metabolism in general, 335 00:14:31,840 --> 00:14:33,640 where else might you want to have 336 00:14:33,640 --> 00:14:36,070 organization of your enzymes? 337 00:14:36,070 --> 00:14:37,990 You might want to have it if you generate 338 00:14:37,990 --> 00:14:39,850 an intermediate in the pathway. 339 00:14:39,850 --> 00:14:41,330 And then there. 340 00:14:41,330 --> 00:14:44,970 It's a branch point for other metabolic pathways. 341 00:14:44,970 --> 00:14:48,970 OK, so there's an intermediate in this pathway that 342 00:14:48,970 --> 00:14:52,140 can go to thiamine biosynthesis to histidine 343 00:14:52,140 --> 00:14:53,745 to tryptophan in biosynthesis. 344 00:14:53,745 --> 00:14:55,120 I'm not going to go through that. 345 00:14:55,120 --> 00:14:56,740 But that would be another place that I 346 00:14:56,740 --> 00:14:59,860 think it's obvious that you could sequester, 347 00:14:59,860 --> 00:15:01,810 under a different set of conditions, 348 00:15:01,810 --> 00:15:04,340 and prevent the other pathways from happening. 349 00:15:04,340 --> 00:15:12,230 So if you have an intermediate branch point, 350 00:15:12,230 --> 00:15:14,500 you can prevent other pathways. 351 00:15:19,340 --> 00:15:26,150 So those two things I think are important. 352 00:15:26,150 --> 00:15:30,910 One of the questions is, do you increase the flux 353 00:15:30,910 --> 00:15:31,960 through the pathway? 354 00:15:31,960 --> 00:15:34,690 OK, so there's been a lot of engineering people. 355 00:15:34,690 --> 00:15:37,240 People really care about this in terms of engineering. 356 00:15:37,240 --> 00:15:41,050 If you want to engineer a metabolic pathway, 357 00:15:41,050 --> 00:15:44,750 should you be linking all your proteins together? 358 00:15:44,750 --> 00:15:47,000 And there have been a lot of papers published. 359 00:15:47,000 --> 00:15:49,070 If you look at bioengineering papers, where 360 00:15:49,070 --> 00:15:52,250 they link all the pathways, all of the enzymes together 361 00:15:52,250 --> 00:15:54,920 in a way, because they want them to cluster, because they 362 00:15:54,920 --> 00:15:58,350 think they're increasing the flux through the pathway. 363 00:15:58,350 --> 00:16:00,110 And so there are some people that 364 00:16:00,110 --> 00:16:03,410 do calculations that show you increase the flux. 365 00:16:03,410 --> 00:16:07,550 Other people do calculations so you don't increase the flux. 366 00:16:07,550 --> 00:16:10,310 So I think, again, this is an area 367 00:16:10,310 --> 00:16:11,762 that I think is very active. 368 00:16:11,762 --> 00:16:13,970 And it's pertinent, because everybody and his brother 369 00:16:13,970 --> 00:16:15,730 is trying to make biofuels. 370 00:16:15,730 --> 00:16:18,290 You'd need to do a lot of engineering 371 00:16:18,290 --> 00:16:20,540 from a lot of enzymes from different places, 372 00:16:20,540 --> 00:16:21,570 putting them together. 373 00:16:21,570 --> 00:16:23,250 How do you make them efficient? 374 00:16:23,250 --> 00:16:28,640 OK, so we asked the question about flux. 375 00:16:28,640 --> 00:16:31,520 And I think, mathematically, people are looking at that. 376 00:16:31,520 --> 00:16:34,440 You need to know a lot about the kinetics of your system. 377 00:16:34,440 --> 00:16:38,190 These systems, there's a lot known about the kinetics. 378 00:16:38,190 --> 00:16:40,580 So and then this goes to the question 379 00:16:40,580 --> 00:16:42,717 of how, what is unstable. 380 00:16:42,717 --> 00:16:44,300 And you need to think about diffusion. 381 00:16:44,300 --> 00:16:48,080 I think this is not so easy to think about this. 382 00:16:48,080 --> 00:16:50,870 But we do need to think about flux through the pathway. 383 00:16:50,870 --> 00:16:53,300 And then the other thing that's interesting in terms 384 00:16:53,300 --> 00:16:57,200 of regulation is it turns out in eukaryotes, where things 385 00:16:57,200 --> 00:17:00,800 are much more regulated than in prokaryotes, 386 00:17:00,800 --> 00:17:03,590 because of the increased complexity of everything. 387 00:17:03,590 --> 00:17:08,240 Almost all of these pathways are organized 388 00:17:08,240 --> 00:17:12,488 on multiple activities on one polypeptide. 389 00:17:12,488 --> 00:17:14,030 That's telling us something, I think, 390 00:17:14,030 --> 00:17:16,893 since we see this over and over and over again. 391 00:17:16,893 --> 00:17:18,560 So there must be some reason to do that. 392 00:17:18,560 --> 00:17:21,800 So for all of these reasons in terms of the purine pathway 393 00:17:21,800 --> 00:17:25,069 this has been sort of a target for people for a long time. 394 00:17:25,069 --> 00:17:27,470 That's one of the reasons I decided to talk about it, 395 00:17:27,470 --> 00:17:30,530 because this was one of the first papers where people were 396 00:17:30,530 --> 00:17:33,820 excited that they thought they had evidence 397 00:17:33,820 --> 00:17:37,610 for this kind of organization in the cell. 398 00:17:37,610 --> 00:17:38,940 Not in the animal. 399 00:17:38,940 --> 00:17:40,260 But in the cell. 400 00:17:40,260 --> 00:17:41,430 OK. 401 00:17:41,430 --> 00:17:43,970 Let's see what I want to say next. 402 00:17:43,970 --> 00:17:47,600 I'm trying to keep this on some kind of a schedule. 403 00:17:47,600 --> 00:17:51,740 OK, so this is the hypothesis. 404 00:17:51,740 --> 00:17:55,050 The hypothesis is that these things 405 00:17:55,050 --> 00:17:57,302 are organized in some way. 406 00:17:57,302 --> 00:17:59,760 And this was taken out of-- probably it was a review paper. 407 00:17:59,760 --> 00:18:02,280 It wasn't taken out of a paper you had to read. 408 00:18:02,280 --> 00:18:03,060 Here's the cell. 409 00:18:03,060 --> 00:18:05,170 That's the nucleus of the cell. 410 00:18:05,170 --> 00:18:06,590 And what do you see. 411 00:18:06,590 --> 00:18:08,400 I think you can see this right. 412 00:18:08,400 --> 00:18:13,870 You see these little dots which they call punctate staining. 413 00:18:13,870 --> 00:18:15,840 So what else do you need to know that they 414 00:18:15,840 --> 00:18:17,280 don't have in this picture that's 415 00:18:17,280 --> 00:18:20,200 really sort of key to thinking about this model. 416 00:18:20,200 --> 00:18:23,640 So here they've just have a bunch of enzymes stuck together 417 00:18:23,640 --> 00:18:25,860 and all in a little ball. 418 00:18:25,860 --> 00:18:31,313 OK, so if you read the paper there was a couple of things. 419 00:18:31,313 --> 00:18:33,230 AUDIENCE: How you're getting the fluorescence? 420 00:18:33,230 --> 00:18:34,470 JOANNE STUBBE: How you're getting them? 421 00:18:34,470 --> 00:18:35,860 AUDIENCE: If it's by effusion or fluorescence. 422 00:18:35,860 --> 00:18:38,490 JOANNE STUBBE: Yeah, so how you're getting the fluorescence 423 00:18:38,490 --> 00:18:39,270 becomes key. 424 00:18:39,270 --> 00:18:41,230 OK, so we're going to talk about that. 425 00:18:41,230 --> 00:18:45,110 How did what was a major way they got the data. 426 00:18:45,110 --> 00:18:47,318 We'll talk about this in a minute in more detail but. 427 00:18:47,318 --> 00:18:49,235 But whenever you're going to use fluorescence, 428 00:18:49,235 --> 00:18:51,770 you have to figure out how to get a probe onto your protein. 429 00:18:51,770 --> 00:18:53,270 So that's like a major focus. 430 00:18:53,270 --> 00:18:56,790 And this again is where chemical biology needs to play a role. 431 00:18:56,790 --> 00:18:59,817 We still need better ways to be able to do this. 432 00:18:59,817 --> 00:19:01,650 You've seen over the course of the semester. 433 00:19:01,650 --> 00:19:04,880 I think in a lot of ways you could potentially do this. 434 00:19:04,880 --> 00:19:06,630 We'll come back to that in a minute. 435 00:19:06,630 --> 00:19:09,810 But if you look at this, what's missing? 436 00:19:09,810 --> 00:19:12,030 And this is something that drove me crazy when 437 00:19:12,030 --> 00:19:14,098 I reviewed the original paper. 438 00:19:14,098 --> 00:19:16,140 AUDIENCE: I just noticed, so you're getting that. 439 00:19:16,140 --> 00:19:19,068 But they didn't stain the membranes really. 440 00:19:19,068 --> 00:19:20,044 There's not a good-- 441 00:19:20,044 --> 00:19:22,484 I mean, you can kind of see the shape of the cell. 442 00:19:22,484 --> 00:19:23,948 But it would be nice to have a clear sort of-- 443 00:19:23,948 --> 00:19:25,670 JOANNE STUBBE: OK, so they might have done that. 444 00:19:25,670 --> 00:19:27,470 Did you look at the supplementary material? 445 00:19:27,470 --> 00:19:29,870 They might have stained the membrane. 446 00:19:29,870 --> 00:19:33,590 OK, so I think everybody would believe you see little blobs. 447 00:19:33,590 --> 00:19:36,470 OK, so what do you need to think about in terms 448 00:19:36,470 --> 00:19:37,445 of the little blob. 449 00:19:37,445 --> 00:19:37,790 AUDIENCE: The size. 450 00:19:37,790 --> 00:19:38,790 JOANNE STUBBE: The size. 451 00:19:38,790 --> 00:19:39,460 Right. 452 00:19:39,460 --> 00:19:41,300 Yeah, so that's one thing. 453 00:19:41,300 --> 00:19:43,940 They don't ever they don't ever talk about this. 454 00:19:43,940 --> 00:19:46,010 They might in some of the very later papers. 455 00:19:46,010 --> 00:19:50,660 But if we know this, we have structures of all the enzymes 456 00:19:50,660 --> 00:19:51,600 in the pathway. 457 00:19:51,600 --> 00:19:54,560 So you could make a guesstimate about how big 458 00:19:54,560 --> 00:19:58,730 these blobs should be, if you had one of each of these. 459 00:19:58,730 --> 00:20:01,300 And these things are huge. 460 00:20:01,300 --> 00:20:05,240 So this would tell you that you would have many, many of these. 461 00:20:05,240 --> 00:20:06,830 This is one thing that I think they 462 00:20:06,830 --> 00:20:11,480 need to do some more thinking about that they could have 463 00:20:11,480 --> 00:20:12,890 many, many of these things. 464 00:20:12,890 --> 00:20:15,140 And then the question is, why would you want 465 00:20:15,140 --> 00:20:17,210 many, many of these things. 466 00:20:17,210 --> 00:20:18,770 And how were they organize? 467 00:20:18,770 --> 00:20:21,380 Are they just sort of randomly organized or are they 468 00:20:21,380 --> 00:20:24,320 really organized in something like that 469 00:20:24,320 --> 00:20:27,000 with this big huge protein in the middle. 470 00:20:27,000 --> 00:20:29,040 That's one of the ones they look at. 471 00:20:29,040 --> 00:20:33,050 FGAM synthase, that has a molecular weight of 150,000, 472 00:20:33,050 --> 00:20:35,040 which is huge for an enzyme. 473 00:20:35,040 --> 00:20:38,180 And so for a long time-- and the catalytic activity-- 474 00:20:38,180 --> 00:20:40,080 my lab has studied that-- is way over here. 475 00:20:40,080 --> 00:20:41,300 And so you have a lot. 476 00:20:41,300 --> 00:20:42,590 Could it be a scaffold. 477 00:20:42,590 --> 00:20:46,380 OK, so that's where that idea actually came from. 478 00:20:46,380 --> 00:20:50,420 So but the hypothesis is that these guys are organized. 479 00:20:50,420 --> 00:20:52,885 And they're under certain growth conditions. 480 00:20:52,885 --> 00:20:53,510 That's the key. 481 00:20:53,510 --> 00:20:56,320 And we'll look at those pictures that 482 00:20:56,320 --> 00:20:59,480 come together if they do this when you need to make purines. 483 00:20:59,480 --> 00:21:01,460 And then they can go apart. 484 00:21:01,460 --> 00:21:05,380 OK, so the key thing, I think, is. 485 00:21:05,380 --> 00:21:07,908 And I wanted to just remind you why 486 00:21:07,908 --> 00:21:09,950 we're spending this time looking at fluorescence. 487 00:21:09,950 --> 00:21:14,150 And we probably should have spent two or three recitations 488 00:21:14,150 --> 00:21:16,400 on fluorescence methods. 489 00:21:16,400 --> 00:21:18,720 But we didn't. 490 00:21:18,720 --> 00:21:20,840 Is that we've seen this many times before. 491 00:21:20,840 --> 00:21:23,820 We've seen stopped-flow fluorescence 492 00:21:23,820 --> 00:21:26,030 in the Rodnina paper, where we were 493 00:21:26,030 --> 00:21:29,750 looking at the kinetics of fidelity of EF-Tu. 494 00:21:29,750 --> 00:21:32,900 And somehow they put a fluorescent probe 495 00:21:32,900 --> 00:21:35,870 onto the piece of tRNA. 496 00:21:35,870 --> 00:21:36,900 That was not trivial. 497 00:21:36,900 --> 00:21:38,450 How you got the probe there. 498 00:21:38,450 --> 00:21:40,167 And that probe could-- 499 00:21:40,167 --> 00:21:41,750 and we'll talk about this in a minute. 500 00:21:41,750 --> 00:21:42,980 But it could change. 501 00:21:42,980 --> 00:21:46,420 It changes when it's in different environments. 502 00:21:46,420 --> 00:21:50,810 And so you can use it as a way to monitor changes. 503 00:21:50,810 --> 00:21:54,080 So reactive oxygen species, we just looked at this. 504 00:21:54,080 --> 00:21:56,270 And I decided to put this up, since we didn't have 505 00:21:56,270 --> 00:21:58,880 the structures up last time. 506 00:21:58,880 --> 00:22:01,110 Fluorescein is one of the dyes that. 507 00:22:01,110 --> 00:22:02,810 This is fluorescein that people use. 508 00:22:02,810 --> 00:22:04,730 This is a version of fluorescein. 509 00:22:04,730 --> 00:22:09,110 But we talked about how do you know 510 00:22:09,110 --> 00:22:14,690 that epidermal growth factor is generating hydrogen peroxide? 511 00:22:14,690 --> 00:22:17,940 OK, so what we need is a sensor of hydrogen peroxide. 512 00:22:17,940 --> 00:22:20,600 So we talked about that last time. 513 00:22:20,600 --> 00:22:23,800 And this is the sensor that people use. 514 00:22:23,800 --> 00:22:24,660 Why did they use it. 515 00:22:24,660 --> 00:22:25,452 We talked about it. 516 00:22:25,452 --> 00:22:26,840 But we didn't have the structure. 517 00:22:26,840 --> 00:22:32,350 So they use the dye acetate of this molecule. 518 00:22:32,350 --> 00:22:33,980 This one they use, the triacetate. 519 00:22:33,980 --> 00:22:38,060 The one that they use in paper was the diacetate. 520 00:22:38,060 --> 00:22:40,760 Anyhow, you need to get the fluorescent probe 521 00:22:40,760 --> 00:22:41,580 into the cell. 522 00:22:41,580 --> 00:22:43,830 So that's something you're going to have to deal with. 523 00:22:43,830 --> 00:22:46,520 And so if you acetylate it, you don't have phenols 524 00:22:46,520 --> 00:22:49,040 or phenolates which might not get through the membrane, 525 00:22:49,040 --> 00:22:50,690 which apparently they don't. 526 00:22:50,690 --> 00:22:52,815 So then when they get in the cell, what do they do? 527 00:22:52,815 --> 00:22:54,200 They hydrolyze, OK. 528 00:22:54,200 --> 00:22:58,370 So what happens is when they hydrolyze, they are now-- 529 00:22:58,370 --> 00:23:00,650 you have these hydroxylated compounds 530 00:23:00,650 --> 00:23:06,790 that are able to be oxidized by an oxidant. 531 00:23:06,790 --> 00:23:09,930 And one of the oxidants that can do this. 532 00:23:09,930 --> 00:23:12,400 And there are others that can do it as well, 533 00:23:12,400 --> 00:23:13,970 is hydrogen peroxide. 534 00:23:13,970 --> 00:23:18,550 So people use this as an indicator of hydrogen peroxide. 535 00:23:18,550 --> 00:23:20,600 But it's not specific. 536 00:23:20,600 --> 00:23:21,450 Yeah? 537 00:23:21,450 --> 00:23:23,854 AUDIENCE: So are they also trapped after that esterase, 538 00:23:23,854 --> 00:23:26,340 like from diffusing that out to the-- 539 00:23:26,340 --> 00:23:27,100 JOANNE STUBBE: No. 540 00:23:27,100 --> 00:23:29,110 I mean, I don't think they diffuse back out, 541 00:23:29,110 --> 00:23:31,480 because I think they're the phenolates. 542 00:23:31,480 --> 00:23:34,450 So I think the diffusion out, like with many of these things, 543 00:23:34,450 --> 00:23:35,440 like if you use-- 544 00:23:35,440 --> 00:23:38,975 lots of times you esterify phosphates 545 00:23:38,975 --> 00:23:40,100 to get them into the cells. 546 00:23:40,100 --> 00:23:41,620 Once they hydrolyze, they charge. 547 00:23:41,620 --> 00:23:44,110 They don't get back out. 548 00:23:44,110 --> 00:23:45,880 So I don't really know. 549 00:23:45,880 --> 00:23:47,440 But that's what I would guess. 550 00:23:47,440 --> 00:23:52,270 So I guess the key thing and the basis for some comments 551 00:23:52,270 --> 00:23:55,600 that I made in class was that we don't really have. 552 00:23:55,600 --> 00:23:58,600 We don't know that this is specific for one 553 00:23:58,600 --> 00:24:00,760 reactive oxygen species. 554 00:24:00,760 --> 00:24:05,360 And so there are lots of people in the chemistry, 555 00:24:05,360 --> 00:24:08,390 biology interface trying to make specific sensors. 556 00:24:08,390 --> 00:24:09,890 OK, that's not easy to do. 557 00:24:09,890 --> 00:24:12,030 The hydrogen peroxide, they're getting better. 558 00:24:12,030 --> 00:24:16,720 In fact, Ting's APEX, which is a peroxidase, sort of similar 559 00:24:16,720 --> 00:24:20,140 to what we talked about with peroxireductions 560 00:24:20,140 --> 00:24:24,640 in the myeloperoxidase can actually 561 00:24:24,640 --> 00:24:28,480 function as a hydrogen peroxide sensor. 562 00:24:28,480 --> 00:24:32,320 So anyhow, what happens is that when it gets oxidized, 563 00:24:32,320 --> 00:24:33,525 it becomes fluorescent. 564 00:24:33,525 --> 00:24:34,525 So it's not fluorescent. 565 00:24:34,525 --> 00:24:35,490 It becomes fluorescent. 566 00:24:35,490 --> 00:24:36,940 So it just gets turned on. 567 00:24:36,940 --> 00:24:39,350 And you can see something, OK? 568 00:24:39,350 --> 00:24:41,350 So that's something we talked about. 569 00:24:43,982 --> 00:24:46,190 In Liz's part of the course, we talked about the fact 570 00:24:46,190 --> 00:24:52,150 that we can watch protein unfolding in the e. 571 00:24:52,150 --> 00:24:53,430 Coli proteasome. 572 00:24:53,430 --> 00:24:54,210 OK. 573 00:24:54,210 --> 00:24:58,660 And what did you look at in the proteasome, clip X clip P? 574 00:24:58,660 --> 00:25:00,660 You looked at titin. 575 00:25:00,660 --> 00:25:03,300 That had a little tryptophan on it. 576 00:25:03,300 --> 00:25:04,930 And tryptophan can absorb. 577 00:25:04,930 --> 00:25:08,950 It's not a very good thing, because it absorbs in the UV. 578 00:25:08,950 --> 00:25:11,920 But tryptophan fluorescence is used a lot. 579 00:25:11,920 --> 00:25:14,640 There are lots of tryptophans, so it's also really hard 580 00:25:14,640 --> 00:25:15,140 to use. 581 00:25:15,140 --> 00:25:17,380 But titin was this little tiny protein. 582 00:25:17,380 --> 00:25:18,850 And it was the only tryptophan. 583 00:25:18,850 --> 00:25:21,910 And they also did experiments with green fluorescent protein, 584 00:25:21,910 --> 00:25:23,950 which is what we're using in this paper. 585 00:25:23,950 --> 00:25:24,700 We remember. 586 00:25:24,700 --> 00:25:26,080 They pull on it. 587 00:25:26,080 --> 00:25:27,850 And you pull and you pull when you pull 588 00:25:27,850 --> 00:25:29,530 and then all of a sudden it unfolds. 589 00:25:29,530 --> 00:25:31,300 And you lose your chromophore. 590 00:25:31,300 --> 00:25:35,380 So you go from the on state to the off state. 591 00:25:35,380 --> 00:25:37,240 So all of these things. 592 00:25:37,240 --> 00:25:38,660 Binding measurements. 593 00:25:38,660 --> 00:25:39,400 You talked about. 594 00:25:39,400 --> 00:25:40,400 You had one problem set. 595 00:25:40,400 --> 00:25:42,817 I don't know whether you guys did that problem set or not. 596 00:25:42,817 --> 00:25:43,570 But there was a-- 597 00:25:46,210 --> 00:25:47,530 what was the calcium sensor? 598 00:25:47,530 --> 00:25:48,490 Does anybody remember? 599 00:25:48,490 --> 00:25:50,070 Anyhow, there was the calcium sensor, 600 00:25:50,070 --> 00:25:52,190 where you were asked in the problem set 601 00:25:52,190 --> 00:25:54,380 for a something or other that you 602 00:25:54,380 --> 00:25:55,910 asked to measure the KD for. 603 00:25:55,910 --> 00:25:58,330 And you can do binding assays. 604 00:25:58,330 --> 00:26:00,730 So fluorescence is an incredibly powerful tool 605 00:26:00,730 --> 00:26:02,470 as is the take home message. 606 00:26:02,470 --> 00:26:04,150 And we've seen it throughout the course. 607 00:26:04,150 --> 00:26:05,890 We just haven't talked about it. 608 00:26:05,890 --> 00:26:08,110 So now the key thing. 609 00:26:08,110 --> 00:26:11,450 And we're going to talk a little bit about fluorescence 610 00:26:11,450 --> 00:26:13,840 at probably a freshman level. 611 00:26:13,840 --> 00:26:16,420 Many of you guys, who were the undergraduates. 612 00:26:16,420 --> 00:26:20,130 You guys, have you done fluorescence experiments? 613 00:26:20,130 --> 00:26:25,000 You haven't done in the lab? 614 00:26:25,000 --> 00:26:27,940 I thought we had two Eureka labs that were fluorescence 615 00:26:27,940 --> 00:26:28,540 oriented. 616 00:26:28,540 --> 00:26:32,340 AUDIENCE: [INAUDIBLE]? 617 00:26:32,340 --> 00:26:33,290 JOANNE STUBBE: No? 618 00:26:33,290 --> 00:26:36,855 AUDIENCE: Yeah, yeah. so we-- 619 00:26:36,855 --> 00:26:39,460 [INTERPOSING VOICES] 620 00:26:39,460 --> 00:26:43,450 JOANNE STUBBE: So doesn't Tim's? 621 00:26:43,450 --> 00:26:45,940 He does sensors to sniff. 622 00:26:45,940 --> 00:26:48,843 I don't know what to sniff, but to sniff something, TNT or-- 623 00:26:48,843 --> 00:26:51,260 AUDIENCE: Right. but we didn't use fluorescence with that. 624 00:26:51,260 --> 00:26:53,660 JOANNE STUBBE: You didn't use fluorescence for that. 625 00:26:53,660 --> 00:26:55,940 OK, or the Tokmakoff lab? 626 00:26:55,940 --> 00:26:58,330 AUDIENCE: The one experiment we did in lab is we 627 00:26:58,330 --> 00:27:03,970 labeled a protein, the green absorbing dye. 628 00:27:03,970 --> 00:27:09,430 And it used laser anisotropy to measure KD rotations. 629 00:27:09,430 --> 00:27:10,678 And so the-- 630 00:27:10,678 --> 00:27:12,720 JOANNE STUBBE: OK, so you guys are experts, then, 631 00:27:12,720 --> 00:27:14,140 on fluorescence. 632 00:27:14,140 --> 00:27:18,870 Well, hopefully you-- anyhow, so one of the questions 633 00:27:18,870 --> 00:27:22,305 is we need to ultimately the key thing for any of this 634 00:27:22,305 --> 00:27:24,180 is we're going to have to have a fluorophore. 635 00:27:24,180 --> 00:27:25,050 So that's it. 636 00:27:25,050 --> 00:27:29,520 So we need the key starting point is a fluorophore 637 00:27:29,520 --> 00:27:32,800 And what are fluorophores. 638 00:27:32,800 --> 00:27:36,370 So you want something that's usually aromatic and large. 639 00:27:36,370 --> 00:27:40,340 It could be-- it could have a lot of nitrogens in it. 640 00:27:40,340 --> 00:27:42,040 Oh, I knew I forgot something. 641 00:27:42,040 --> 00:27:44,730 So there's a book called, Molecular Probes. 642 00:27:44,730 --> 00:27:49,098 OK so I gave you a handout on fluorescence. 643 00:27:49,098 --> 00:27:51,390 I forgot to bring the book, if anybody wants to see it. 644 00:27:51,390 --> 00:27:53,160 This book is worth its weight in gold 645 00:27:53,160 --> 00:27:54,673 if you're a chemical biologist. 646 00:27:54,673 --> 00:27:56,340 Because this has everything in the world 647 00:27:56,340 --> 00:27:57,840 you need to know about fluorescence. 648 00:27:57,840 --> 00:27:59,760 It's described in a thoughtful way. 649 00:27:59,760 --> 00:28:02,570 They sell all the probes. 650 00:28:02,570 --> 00:28:04,860 If you want to do something to tweak something, 651 00:28:04,860 --> 00:28:07,210 they'll help you do all of that. 652 00:28:07,210 --> 00:28:10,200 So this book, this is molecular probes book. 653 00:28:10,200 --> 00:28:11,350 I think it's online now. 654 00:28:11,350 --> 00:28:13,960 I have a copy that's five years old. 655 00:28:13,960 --> 00:28:15,670 I use it a lot. 656 00:28:15,670 --> 00:28:17,010 It's a really important book. 657 00:28:17,010 --> 00:28:19,583 And I got this out of the book. 658 00:28:19,583 --> 00:28:21,000 And it just shows you in the book, 659 00:28:21,000 --> 00:28:23,210 they have all these pictures of these fluorophores. 660 00:28:23,210 --> 00:28:27,690 So they're just big, huge, greasy molecules. 661 00:28:27,690 --> 00:28:30,170 You have to worry about solubility a lot of the time. 662 00:28:30,170 --> 00:28:32,910 So you have to stick sulfates, or something 663 00:28:32,910 --> 00:28:35,730 that ends up making it soluble. 664 00:28:35,730 --> 00:28:38,380 So that's going to be a key thing. 665 00:28:38,380 --> 00:28:40,380 So we need to have a fluorophore. 666 00:28:40,380 --> 00:28:43,530 And we have many options that we can buy these things. 667 00:28:43,530 --> 00:28:45,810 OK, so what's this? 668 00:28:45,810 --> 00:28:47,860 OK, so what we want to think about is this. 669 00:28:47,860 --> 00:28:51,720 So in your, the latest version of your handouts, 670 00:28:51,720 --> 00:28:53,990 I've written down what I'm going to say. 671 00:28:53,990 --> 00:28:57,980 Butt it's pretty simple for-- 672 00:29:01,870 --> 00:29:06,700 I'm talking about this in a pretty simplified viewpoint. 673 00:29:06,700 --> 00:29:09,240 But what we're going to see is these fluorophores 674 00:29:09,240 --> 00:29:12,600 are going to allow us to. 675 00:29:12,600 --> 00:29:14,550 They allow us to do assays. 676 00:29:14,550 --> 00:29:18,510 I'm going to show you a quick example of that. 677 00:29:18,510 --> 00:29:21,765 That is you can have something that is. 678 00:29:21,765 --> 00:29:24,652 You can have a molecule that is quenched, 679 00:29:24,652 --> 00:29:26,110 so you have a quencher on one side. 680 00:29:26,110 --> 00:29:26,490 I'll show you. 681 00:29:26,490 --> 00:29:28,410 And I'll show you the way the quenching comes from, 682 00:29:28,410 --> 00:29:30,077 something fluorescent on the other side. 683 00:29:30,077 --> 00:29:31,080 You can't see anything. 684 00:29:31,080 --> 00:29:32,530 You cut it in half. 685 00:29:32,530 --> 00:29:33,930 It could be a protease. 686 00:29:33,930 --> 00:29:35,400 It could be a nuclease. 687 00:29:35,400 --> 00:29:36,600 The quencher goes away. 688 00:29:36,600 --> 00:29:39,210 And you see fluorescence. 689 00:29:39,210 --> 00:29:41,820 You could have a sensor for metal binding, which 690 00:29:41,820 --> 00:29:42,720 Liz talked about. 691 00:29:42,720 --> 00:29:44,525 So you have two fluorophores. 692 00:29:44,525 --> 00:29:45,900 OK, you've got to figure out what 693 00:29:45,900 --> 00:29:47,190 the right fluorophores are. 694 00:29:47,190 --> 00:29:48,060 Something binds. 695 00:29:48,060 --> 00:29:49,780 They change confirmation. 696 00:29:49,780 --> 00:29:52,110 And they change confirmation in some way 697 00:29:52,110 --> 00:29:56,250 that you can actually detect a shift in the wavelength. 698 00:29:56,250 --> 00:29:57,320 And then you're looking. 699 00:29:57,320 --> 00:29:59,070 In our case, we're just sticking something 700 00:29:59,070 --> 00:30:00,927 on the end to see something. 701 00:30:00,927 --> 00:30:02,510 You were making a protein fluorescent. 702 00:30:02,510 --> 00:30:04,520 That's all we're doing. 703 00:30:04,520 --> 00:30:06,510 So you can use it for assays. 704 00:30:06,510 --> 00:30:08,200 You can use it for FRET. 705 00:30:08,200 --> 00:30:09,980 And in the current-- 706 00:30:09,980 --> 00:30:11,610 so you can measure distances. 707 00:30:11,610 --> 00:30:14,520 We're not going to go into that. 708 00:30:14,520 --> 00:30:16,350 But any of you that are interested 709 00:30:16,350 --> 00:30:18,330 in the current version of the handout, 710 00:30:18,330 --> 00:30:21,900 I have sort of short tutorial on what FRET is 711 00:30:21,900 --> 00:30:24,700 and where you should go to look this up. 712 00:30:24,700 --> 00:30:28,380 And then we just basically have a fluorescent tag. 713 00:30:28,380 --> 00:30:30,673 OK, and we'll come back and talk about the tag. 714 00:30:30,673 --> 00:30:32,340 We already talked about the fact that we 715 00:30:32,340 --> 00:30:35,157 have green fluorescent protein, red fluorescent protein tags. 716 00:30:35,157 --> 00:30:37,240 But we'll come back and talk about the other tags. 717 00:30:37,240 --> 00:30:39,510 So we have a fluorophore And so what 718 00:30:39,510 --> 00:30:43,600 does that mean in terms of what's going on. 719 00:30:43,600 --> 00:30:46,890 So you have your molecule. 720 00:30:46,890 --> 00:30:54,170 And your molecule has a ground state, 721 00:30:54,170 --> 00:30:56,520 which we'll call this S0. 722 00:30:56,520 --> 00:30:57,690 This is the ground state. 723 00:30:57,690 --> 00:30:59,910 And you have many vibrational modes. 724 00:30:59,910 --> 00:31:02,190 And you have this big huge fluorophore 725 00:31:02,190 --> 00:31:05,160 that can absorb your electron. 726 00:31:05,160 --> 00:31:08,960 And your fluorophore can't absorb a photon. 727 00:31:08,960 --> 00:31:11,150 And so what happens is. 728 00:31:11,150 --> 00:31:16,270 So we're going to have excitation 729 00:31:16,270 --> 00:31:20,220 with a photon in a certain way, in a wavelength that 730 00:31:20,220 --> 00:31:22,810 can be absorbed by the electron in your molecule 731 00:31:22,810 --> 00:31:28,960 to the excited state, which they call S1. 732 00:31:28,960 --> 00:31:33,210 And so you can have your electron 733 00:31:33,210 --> 00:31:36,240 going to an excited state. 734 00:31:36,240 --> 00:31:38,940 And we have a wavelength of light when that happens. 735 00:31:38,940 --> 00:31:41,280 And that depends on the structure of your molecule. 736 00:31:41,280 --> 00:31:44,430 So you don't want to be in the UV region. 737 00:31:44,430 --> 00:31:46,620 You want to be out in the region where 738 00:31:46,620 --> 00:31:48,630 you have less interference. 739 00:31:48,630 --> 00:31:50,100 And so that's the key game you have 740 00:31:50,100 --> 00:31:53,190 to play to get into that region in the visible. 741 00:31:53,190 --> 00:31:55,810 You really have to put a lot of stuff on here. 742 00:31:55,810 --> 00:31:57,960 You just can't make a small little molecule 743 00:31:57,960 --> 00:32:00,560 that absorbs at 600 nanometers. 744 00:32:00,560 --> 00:32:01,810 So that's part of the problem. 745 00:32:01,810 --> 00:32:04,510 So you're making big things of necessity, 746 00:32:04,510 --> 00:32:07,360 so you can actually see something happen. 747 00:32:07,360 --> 00:32:09,750 And so then what happens under those conditions. 748 00:32:09,750 --> 00:32:18,240 So we're going to have the excitation wavelength of light 749 00:32:18,240 --> 00:32:19,690 at a certain lambda max. 750 00:32:19,690 --> 00:32:20,197 You absorb. 751 00:32:20,197 --> 00:32:21,280 It's just like absorption. 752 00:32:21,280 --> 00:32:23,650 You have a certain wavelength that it 753 00:32:23,650 --> 00:32:25,300 absorbs more frequently. 754 00:32:25,300 --> 00:32:31,860 Then what happens in the excited state on a very fast timescale, 755 00:32:31,860 --> 00:32:33,130 you lose energy. 756 00:32:33,130 --> 00:32:37,080 OK, so under these conditions, you're doing a relaxation. 757 00:32:40,638 --> 00:32:41,930 And then we'll see in a minute. 758 00:32:41,930 --> 00:32:44,740 I'll talk about what are the mechanisms of relaxation. 759 00:32:44,740 --> 00:32:45,720 But that can tell you. 760 00:32:45,720 --> 00:32:49,770 You can use those relaxation mechanisms in a different way 761 00:32:49,770 --> 00:32:53,030 to design your fluorescent experiments. 762 00:32:53,030 --> 00:32:56,880 So what you see in this cartoon is 763 00:32:56,880 --> 00:33:01,560 that you're relaxing on a very fast timescale. 764 00:33:01,560 --> 00:33:06,320 And physical chemistry has told us that to see fluorescence, 765 00:33:06,320 --> 00:33:07,980 it needs to go down. 766 00:33:07,980 --> 00:33:09,510 So these are the vibrational modes. 767 00:33:09,510 --> 00:33:14,400 So you're exciting your electron electronically 768 00:33:14,400 --> 00:33:16,800 and vibrationally. 769 00:33:16,800 --> 00:33:20,100 And then you need to go down in vibrations. 770 00:33:20,100 --> 00:33:21,810 You're losing energy somehow. 771 00:33:21,810 --> 00:33:23,030 What happens to that energy? 772 00:33:23,030 --> 00:33:25,460 OK, we can talk about what can happen to that energy. 773 00:33:25,460 --> 00:33:30,000 And when it gets to the lowest level of the excited state, 774 00:33:30,000 --> 00:33:31,340 you have fluorescence. 775 00:33:31,340 --> 00:33:36,750 OK, and so that also happens on a pretty fast timescale. 776 00:33:36,750 --> 00:33:39,060 So the key thing here. 777 00:33:39,060 --> 00:33:40,390 So when you get to the lowest. 778 00:33:40,390 --> 00:33:47,410 So this is the lowest level, it fluoresces. 779 00:33:47,410 --> 00:33:50,990 And so this is where the photon emits. 780 00:33:50,990 --> 00:34:01,520 OK, so the photon wavelength for emission or h nu emission. 781 00:34:01,520 --> 00:34:05,553 And the key thing that you've probably heard about, again, 782 00:34:05,553 --> 00:34:07,220 when you were introduced to fluorescence 783 00:34:07,220 --> 00:34:11,420 is because you're losing energy here, 784 00:34:11,420 --> 00:34:12,920 what happens to the energy? 785 00:34:12,920 --> 00:34:15,000 You're going to a longer wavelength. 786 00:34:15,000 --> 00:34:19,580 OK, so the excitation and the emission wavelengths 787 00:34:19,580 --> 00:34:20,239 are distinct. 788 00:34:20,239 --> 00:34:22,350 And that's called the stoke shift. 789 00:34:22,350 --> 00:34:26,300 So it's the wavelength of excitation vs. the wavelength 790 00:34:26,300 --> 00:34:27,030 of emissions. 791 00:34:27,030 --> 00:34:32,250 So you have a stokes shift, which 792 00:34:32,250 --> 00:34:40,260 is the wavelength of excitation minus the wavelength 793 00:34:40,260 --> 00:34:42,020 of emission. 794 00:34:42,020 --> 00:34:43,787 And so you need to look at molecules. 795 00:34:43,787 --> 00:34:45,120 People have spent a lot of time. 796 00:34:45,120 --> 00:34:48,179 You saw those 25 lists of things where 797 00:34:48,179 --> 00:34:51,030 people have designed things that actually 798 00:34:51,030 --> 00:34:53,469 work quite effectively. 799 00:34:53,469 --> 00:34:57,450 OK, and so then the question is you losing energy. 800 00:34:57,450 --> 00:34:59,860 You are always going to be at longer wavelengths. 801 00:34:59,860 --> 00:35:02,370 OK, so that's good, that makes it easier to see, 802 00:35:02,370 --> 00:35:07,500 because there aren't that many things inside the cell that 803 00:35:07,500 --> 00:35:09,390 give you a background, which is what you need 804 00:35:09,390 --> 00:35:11,670 to worry about in all of the experiments 805 00:35:11,670 --> 00:35:13,460 you're doing inside the cell. 806 00:35:13,460 --> 00:35:17,680 The brightness, we'll come back to that in a minute. 807 00:35:17,680 --> 00:35:20,390 So what kinds of models can give you. 808 00:35:20,390 --> 00:35:22,860 What kinds of mechanisms are there 809 00:35:22,860 --> 00:35:27,420 for relaxation of the excited state. 810 00:35:37,180 --> 00:35:39,670 And so there are a number of mechanisms 811 00:35:39,670 --> 00:35:41,980 that can be involved. 812 00:35:41,980 --> 00:35:46,630 And one is, again, non-radiative relaxation. 813 00:35:52,690 --> 00:35:54,740 And how does that happen? 814 00:35:54,740 --> 00:35:57,080 So you're changing vibrational modes. 815 00:35:57,080 --> 00:35:59,830 And when you're in the excited state, if you're in solution, 816 00:35:59,830 --> 00:36:03,070 you have interactions with solvent or other molecules, 817 00:36:03,070 --> 00:36:05,950 all of which can affect this kind of transition. 818 00:36:05,950 --> 00:36:09,340 If you're in the active site, there can be other things. 819 00:36:09,340 --> 00:36:15,450 So the key here is the environment. 820 00:36:15,450 --> 00:36:18,850 And again, it could be solvent. 821 00:36:18,850 --> 00:36:22,880 It could be protein. 822 00:36:22,880 --> 00:36:24,940 And the only way you can tell is by actually 823 00:36:24,940 --> 00:36:28,930 looking at the fluorophore on your molecule to end up 824 00:36:28,930 --> 00:36:32,750 seeing what you end up seeing. 825 00:36:32,750 --> 00:36:36,640 OK, so a second way that you can see. 826 00:36:36,640 --> 00:36:38,620 And you probably saw this in your introductory. 827 00:36:38,620 --> 00:36:39,255 Yeah? 828 00:36:39,255 --> 00:36:40,838 AUDIENCE: So what would be an example? 829 00:36:40,838 --> 00:36:42,760 Like, if a unit of the energy being released 830 00:36:42,760 --> 00:36:45,679 is a photon in one case for non-radiative, 831 00:36:45,679 --> 00:36:49,900 what's the unit of energy? 832 00:36:49,900 --> 00:36:51,770 JOANNE STUBBE: What is the unit of energy? 833 00:36:51,770 --> 00:36:56,260 So energy, heat is one way that you lose all of this. 834 00:36:56,260 --> 00:36:57,590 So it's vibrational energy. 835 00:36:57,590 --> 00:36:59,920 I would say, it's mostly heat. 836 00:36:59,920 --> 00:37:06,910 So you're changing excitation levels somehow. 837 00:37:06,910 --> 00:37:08,710 And the beauty of fluorescence. 838 00:37:08,710 --> 00:37:11,260 And this is the key to the sensitivity 839 00:37:11,260 --> 00:37:14,710 is you're not doing anything to your molecule. 840 00:37:14,710 --> 00:37:17,570 So your electrons got excited. 841 00:37:17,570 --> 00:37:20,070 They give off a little heat or whatever. 842 00:37:20,070 --> 00:37:22,510 They somehow change a little bit. 843 00:37:22,510 --> 00:37:25,438 And then they go back down to the ground state again. 844 00:37:25,438 --> 00:37:26,410 So what can you do? 845 00:37:26,410 --> 00:37:27,790 You can excite them again. 846 00:37:27,790 --> 00:37:31,570 So this can happen over and over and over again, 847 00:37:31,570 --> 00:37:35,110 unless the molecule in the excited state 848 00:37:35,110 --> 00:37:36,520 becomes destroyed. 849 00:37:36,520 --> 00:37:39,370 So that's called photo bleaching. 850 00:37:39,370 --> 00:37:40,720 So the key thing here. 851 00:37:40,720 --> 00:37:46,290 And this is, I think, this ability to recycle 852 00:37:46,290 --> 00:37:47,980 is the key to sensitivity. 853 00:37:50,830 --> 00:37:54,710 But again, I haven't used fluorescence inside the cell. 854 00:37:54,710 --> 00:37:57,502 I've never done this myself, experimentally. 855 00:37:57,502 --> 00:37:58,460 So I don't really know. 856 00:37:58,460 --> 00:38:01,140 But you hear about photo bleaching all the time. 857 00:38:01,140 --> 00:38:03,020 So I think this is not a trivial thing 858 00:38:03,020 --> 00:38:04,800 that you can just blow off. 859 00:38:04,800 --> 00:38:05,570 It would be nice. 860 00:38:05,570 --> 00:38:09,800 But what you're doing is you're using the same excitation. 861 00:38:09,800 --> 00:38:12,380 And then loss and excitation and loss 862 00:38:12,380 --> 00:38:13,620 over and over and over again. 863 00:38:13,620 --> 00:38:17,870 And so it provides a much more sensitive assay 864 00:38:17,870 --> 00:38:22,620 than what you normally see for something like absorption. 865 00:38:22,620 --> 00:38:24,797 OK, so let's see. 866 00:38:24,797 --> 00:38:25,880 There was one other thing. 867 00:38:25,880 --> 00:38:30,830 Oh, so we talked about this mechanism, non radiative 868 00:38:30,830 --> 00:38:32,600 relaxation. 869 00:38:32,600 --> 00:38:35,360 How else could you relax? 870 00:38:35,360 --> 00:38:39,080 You can go from a singlet state to a triplet state. 871 00:38:39,080 --> 00:38:40,400 OK, I'm not going to talk. 872 00:38:40,400 --> 00:38:42,560 But intersystem crossing, yeah. 873 00:38:42,560 --> 00:38:51,490 So you can go from the singlet excited state 874 00:38:51,490 --> 00:38:53,015 to the triplet state. 875 00:38:53,015 --> 00:38:54,390 I'm not going to talk about this. 876 00:38:54,390 --> 00:38:57,500 But the triplet state then can phosphoresce. 877 00:39:01,800 --> 00:39:04,560 We're not going to be discussing that at all. 878 00:39:04,560 --> 00:39:06,960 But that's one possibility. 879 00:39:06,960 --> 00:39:10,110 We just talked about the fact that you 880 00:39:10,110 --> 00:39:13,590 can have something in there that quenches the fluorescence. 881 00:39:13,590 --> 00:39:17,860 It interacts with something in a distance dependent fashion. 882 00:39:17,860 --> 00:39:22,220 And that, again, affects the intensity of your fluorescence. 883 00:39:22,220 --> 00:39:26,850 So you also have reaction with the second molecule. 884 00:39:33,360 --> 00:39:34,660 And that can become. 885 00:39:34,660 --> 00:39:36,370 And it could be good or bad. 886 00:39:36,370 --> 00:39:40,450 If it reacts with oxygen, what happens is oxygen, 887 00:39:40,450 --> 00:39:42,992 the energy is immediately transferred to the oxygen. 888 00:39:42,992 --> 00:39:44,950 So that's why in many fluorescence experiments, 889 00:39:44,950 --> 00:39:47,050 you remove oxygen from all of your samples. 890 00:39:47,050 --> 00:39:48,530 It acts as a quencher. 891 00:39:48,530 --> 00:39:49,330 So you have. 892 00:39:49,330 --> 00:39:54,660 And it could be oxygen, which acts as a quencher. 893 00:39:54,660 --> 00:39:58,030 Or it could be another fluorophore. 894 00:39:58,030 --> 00:39:58,880 In which case. 895 00:39:58,880 --> 00:40:02,330 and if everything is set up correctly, 896 00:40:02,330 --> 00:40:04,990 you can get the energy to shift the energy of emission 897 00:40:04,990 --> 00:40:06,820 can get shifted to longer wavelengths. 898 00:40:06,820 --> 00:40:09,250 So that's what FRET is all about. 899 00:40:09,250 --> 00:40:10,780 OK, so it could not. 900 00:40:10,780 --> 00:40:14,200 A second molecule could be another fluorophore. 901 00:40:14,200 --> 00:40:18,880 OK, so those are sort of ways that you can relax. 902 00:40:18,880 --> 00:40:22,600 And then you can set up different kinds of experiments, 903 00:40:22,600 --> 00:40:25,300 depending upon what the objective is 904 00:40:25,300 --> 00:40:28,090 of using fluorescence. 905 00:40:28,090 --> 00:40:31,940 So I've written this out in more detail. 906 00:40:31,940 --> 00:40:35,720 And for those of you who want to look at FRET, 907 00:40:35,720 --> 00:40:37,020 I've defined FRET. 908 00:40:37,020 --> 00:40:39,550 I've given you the equations. 909 00:40:39,550 --> 00:40:42,620 And people use this quite a bit inside the cell. 910 00:40:42,620 --> 00:40:44,310 You need to study this. 911 00:40:44,310 --> 00:40:46,480 There are a lot of issues associated with it 912 00:40:46,480 --> 00:40:48,458 that you need to think about. 913 00:40:48,458 --> 00:40:49,250 And I'll come back. 914 00:40:49,250 --> 00:40:50,990 You need to think about. 915 00:40:50,990 --> 00:40:52,060 It's not. 916 00:40:52,060 --> 00:40:55,540 There are a lot of constants that 917 00:40:55,540 --> 00:40:58,180 determine the rate constant for your FRET, OK. 918 00:40:58,180 --> 00:41:01,120 And so you just you need to think about all these constants 919 00:41:01,120 --> 00:41:05,770 to be able to interpret the data in a thoughtful way. 920 00:41:05,770 --> 00:41:08,740 And I've given you a tutorial that I 921 00:41:08,740 --> 00:41:12,330 felt was pretty good that I get off the web that 922 00:41:12,330 --> 00:41:14,640 just shows what FRET is. 923 00:41:14,640 --> 00:41:19,530 And that we have many, many dyes that we can measure distances 924 00:41:19,530 --> 00:41:22,678 from 10 to 100 Angstroms using FRET. 925 00:41:22,678 --> 00:41:23,720 That's not in this paper. 926 00:41:23,720 --> 00:41:24,420 So I didn't. 927 00:41:24,420 --> 00:41:25,888 And this just sort of is a cartoon 928 00:41:25,888 --> 00:41:27,180 of what I was just telling you. 929 00:41:27,180 --> 00:41:30,730 So here, you might have an interaction. 930 00:41:30,730 --> 00:41:35,100 But if you cut it, the interaction could be gone. 931 00:41:35,100 --> 00:41:38,340 Here, you might have no interaction. 932 00:41:38,340 --> 00:41:41,940 But when some small molecule binds, you see an interaction. 933 00:41:41,940 --> 00:41:44,350 And you can pick this up using fluorescence changes. 934 00:41:44,350 --> 00:41:49,020 OK, so people do these kinds of experiments all the time. 935 00:41:49,020 --> 00:41:51,840 And this kind of an assay is extremely-- 936 00:41:51,840 --> 00:41:54,160 there are two kinds of assays that one does. 937 00:41:54,160 --> 00:41:56,580 So if you work in a pharmaceutical company, 938 00:41:56,580 --> 00:41:57,785 people do this all the time. 939 00:41:57,785 --> 00:41:59,160 They want a very sensitive assay. 940 00:41:59,160 --> 00:42:00,960 Everybody uses fluorescence. 941 00:42:00,960 --> 00:42:02,970 They might use an assay like this, where you 942 00:42:02,970 --> 00:42:04,780 go from nothing to something. 943 00:42:04,780 --> 00:42:07,530 OK, so you have high sensitivity. 944 00:42:07,530 --> 00:42:10,830 And the other thing they use is, which I gave you 945 00:42:10,830 --> 00:42:13,560 in your handout, is fluorescence polarization, which I'm not 946 00:42:13,560 --> 00:42:14,980 going to be talking about. 947 00:42:14,980 --> 00:42:17,520 But those are the two major methods 948 00:42:17,520 --> 00:42:19,300 that people develop assays around 949 00:42:19,300 --> 00:42:21,280 in the pharmaceutical industry. 950 00:42:21,280 --> 00:42:23,830 So fluorescence is here to stay. 951 00:42:23,830 --> 00:42:26,180 We still need better tools. 952 00:42:26,180 --> 00:42:28,470 It can be quantitative. 953 00:42:28,470 --> 00:42:30,420 You can measure a quantum efficiency 954 00:42:30,420 --> 00:42:33,510 of the electron, light, that's involved 955 00:42:33,510 --> 00:42:35,910 in the excitation and the photon that's 956 00:42:35,910 --> 00:42:37,080 involved in the emission. 957 00:42:37,080 --> 00:42:40,590 If it's 100 percent efficient, then you're quantum efficiency 958 00:42:40,590 --> 00:42:41,910 is 1 anyhow. 959 00:42:41,910 --> 00:42:46,300 So you have a whole range of quantum efficiencies. 960 00:42:46,300 --> 00:42:53,120 OK, so now what I want to do is we're late. 961 00:42:53,120 --> 00:42:58,710 But we'll at least get to the other sources telling you 962 00:42:58,710 --> 00:42:59,700 what I just told you. 963 00:42:59,700 --> 00:43:03,478 OK, so I want to just introduce to you 964 00:43:03,478 --> 00:43:05,520 some of the issues that we're going to be facing. 965 00:43:05,520 --> 00:43:07,740 And we are going to talk about this in class, 966 00:43:07,740 --> 00:43:10,050 probably Monday or on Wednesday morning, OK. 967 00:43:10,050 --> 00:43:11,940 So I'll extend this in class. 968 00:43:11,940 --> 00:43:15,390 But they've attached green fluorescent protein 969 00:43:15,390 --> 00:43:16,480 to all of these things. 970 00:43:16,480 --> 00:43:18,360 So this is issue number one. 971 00:43:18,360 --> 00:43:20,460 What should they have done in these papers 972 00:43:20,460 --> 00:43:22,850 that they didn't do? 973 00:43:22,850 --> 00:43:24,450 If you read the paper carefully. 974 00:43:24,450 --> 00:43:27,580 I mean, it's hard to read a science paper, 975 00:43:27,580 --> 00:43:29,910 because all the key pieces of data 976 00:43:29,910 --> 00:43:32,680 are in supplementary information. 977 00:43:32,680 --> 00:43:33,778 So they made a few. 978 00:43:33,778 --> 00:43:35,820 In all of these, I can't remember what they made. 979 00:43:35,820 --> 00:43:38,070 But they made fusion proteins, right? 980 00:43:38,070 --> 00:43:40,110 So here, you have a purine enzyme. 981 00:43:40,110 --> 00:43:44,770 And here we have some kind of fluorescent protein. 982 00:43:44,770 --> 00:43:47,050 So that's the probe they're using. 983 00:43:47,050 --> 00:43:49,035 OK, so what's wrong with that, with the way 984 00:43:49,035 --> 00:43:50,160 they did their experiments? 985 00:43:50,160 --> 00:43:55,160 Can anybody look at the details of what's going on? 986 00:43:58,200 --> 00:43:59,610 So what, if you made this fusion, 987 00:43:59,610 --> 00:44:00,630 what would be the first thing you 988 00:44:00,630 --> 00:44:01,950 would do with a fusion protein? 989 00:44:01,950 --> 00:44:03,351 AUDIENCE: My first thought would be, 990 00:44:03,351 --> 00:44:05,476 if it changes the activity of the original protein. 991 00:44:05,476 --> 00:44:06,775 GFP's a very large [INAUDIBLE]. 992 00:44:06,775 --> 00:44:07,650 JOANNE STUBBE: Right. 993 00:44:07,650 --> 00:44:08,180 Exactly. 994 00:44:08,180 --> 00:44:10,080 GFP I'm going to show you in a second. 995 00:44:10,080 --> 00:44:11,910 I think I can show you this in a second. 996 00:44:11,910 --> 00:44:13,980 These are just the ways they were looking. 997 00:44:13,980 --> 00:44:15,690 But you have all these probes. 998 00:44:15,690 --> 00:44:16,980 GFP, it's over here. 999 00:44:16,980 --> 00:44:19,110 These are the organic dyes. 1000 00:44:19,110 --> 00:44:20,040 Here's an antibody. 1001 00:44:20,040 --> 00:44:21,040 We'll come back to that. 1002 00:44:21,040 --> 00:44:23,500 So GFP is big. 1003 00:44:23,500 --> 00:44:26,160 So does it change activity? 1004 00:44:26,160 --> 00:44:27,570 They didn't assay that. 1005 00:44:27,570 --> 00:44:28,863 To me, that's mind boggling. 1006 00:44:28,863 --> 00:44:30,280 OK, because I've dealt with these. 1007 00:44:30,280 --> 00:44:32,490 I know these proteins, that one protein there. 1008 00:44:32,490 --> 00:44:33,990 So two of them they're dealing with. 1009 00:44:33,990 --> 00:44:35,573 One of them is a trifuctional protein. 1010 00:44:35,573 --> 00:44:37,290 The other one's 150 kilodaltons. 1011 00:44:37,290 --> 00:44:40,740 These proteins are not easy to deal with, OK. 1012 00:44:40,740 --> 00:44:43,090 So to me, this is a key thing. 1013 00:44:43,090 --> 00:44:45,570 So this goes back to the Marcotte paper, 1014 00:44:45,570 --> 00:44:47,790 where he's saying, well, I mean, maybe these things 1015 00:44:47,790 --> 00:44:49,188 don't express very well. 1016 00:44:49,188 --> 00:44:49,980 And they aggregate. 1017 00:44:49,980 --> 00:44:50,670 They don't fold. 1018 00:44:50,670 --> 00:44:54,840 We saw how complicated the folding process is. 1019 00:44:54,840 --> 00:44:56,460 What is the second thing? 1020 00:44:56,460 --> 00:44:58,880 How did they get the proteins into the cell? 1021 00:45:01,972 --> 00:45:02,680 How did they get? 1022 00:45:02,680 --> 00:45:04,263 They don't get proteins into the cell. 1023 00:45:04,263 --> 00:45:06,050 How did they get? 1024 00:45:06,050 --> 00:45:09,445 Yeah, how did they get GFP constructs into the cell? 1025 00:45:09,445 --> 00:45:10,820 AUDIENCE: Transient transfection? 1026 00:45:10,820 --> 00:45:14,070 JOANNE STUBBE: Yeah, transient transfection, 1027 00:45:14,070 --> 00:45:15,840 what is the issue there? 1028 00:45:19,450 --> 00:45:22,345 Without going into details, but what's the issue? 1029 00:45:22,345 --> 00:45:26,305 AUDIENCE: Like, when the cell's normal mechanism, 1030 00:45:26,305 --> 00:45:29,770 like the cell's own enzymes maybe-- 1031 00:45:29,770 --> 00:45:32,210 JOANNE STUBBE: So you do have a normal-- 1032 00:45:32,210 --> 00:45:33,460 you do have the normal enzyme. 1033 00:45:33,460 --> 00:45:37,050 They didn't make any effort to knock out the purine enzymes. 1034 00:45:37,050 --> 00:45:43,940 OK, but I think the key thing with transient transfection 1035 00:45:43,940 --> 00:45:46,578 is the levels. 1036 00:45:46,578 --> 00:45:48,620 First of all, a lot of cells don't have anything. 1037 00:45:48,620 --> 00:45:49,960 But then you don't care about that, 1038 00:45:49,960 --> 00:45:51,440 because you don't look at them, because they're not 1039 00:45:51,440 --> 00:45:52,240 fluorescent. 1040 00:45:52,240 --> 00:45:56,997 OK, but do you think the levels are important. 1041 00:45:56,997 --> 00:45:58,830 I think the levels are incredibly important. 1042 00:45:58,830 --> 00:46:01,130 So the question is 100-fold, 1,000 1043 00:46:01,130 --> 00:46:04,850 fold over the endogenous levels. 1044 00:46:04,850 --> 00:46:07,018 And so to me, the first experiments 1045 00:46:07,018 --> 00:46:09,560 I would have done before I did any of these other experiments 1046 00:46:09,560 --> 00:46:10,940 is I would have looked at. 1047 00:46:10,940 --> 00:46:13,700 You might have chosen the trifunctional protein, 1048 00:46:13,700 --> 00:46:18,900 which they did, because it has activities 2, 3, and 5. 1049 00:46:18,900 --> 00:46:21,215 And this other big huge protein, which-- 1050 00:46:21,215 --> 00:46:24,350 so these are the proteins they focus on is activity 4. 1051 00:46:24,350 --> 00:46:25,790 So 4 is huge. 1052 00:46:25,790 --> 00:46:27,290 You might think it could function 1053 00:46:27,290 --> 00:46:30,960 as a scaffolding protein to interact with activities 2, 3, 1054 00:46:30,960 --> 00:46:31,460 5. 1055 00:46:31,460 --> 00:46:33,380 All of that's totally reasonable. 1056 00:46:33,380 --> 00:46:37,620 OK, but they didn't deal with those issues. 1057 00:46:37,620 --> 00:46:42,050 So you need to figure out how to attach 1058 00:46:42,050 --> 00:46:43,860 something that's fluorescent. 1059 00:46:43,860 --> 00:46:45,950 So one way is genetically. 1060 00:46:45,950 --> 00:46:47,800 OK, and we've seen this. 1061 00:46:47,800 --> 00:46:51,665 So we're just fusing GFP onto the protein of interest. 1062 00:46:54,580 --> 00:46:57,730 Another way in this paper, also, and you mentioned that, 1063 00:46:57,730 --> 00:47:00,180 is they were using endogenous antibodies. 1064 00:47:00,180 --> 00:47:04,540 OK, so antibodies can't get into cells. 1065 00:47:04,540 --> 00:47:05,610 So how do you assay this? 1066 00:47:05,610 --> 00:47:08,820 So these are also tough experiments. 1067 00:47:08,820 --> 00:47:12,910 So somehow you fix the cells. 1068 00:47:12,910 --> 00:47:14,490 So they aren't falling apart when 1069 00:47:14,490 --> 00:47:18,030 you're trying to perturb the cell to allow 1070 00:47:18,030 --> 00:47:20,640 the antibodies to get in. 1071 00:47:20,640 --> 00:47:22,260 And then you permeabilize the cells. 1072 00:47:22,260 --> 00:47:24,000 Have any of you ever done that? 1073 00:47:24,000 --> 00:47:25,710 I've done it in yeast. 1074 00:47:25,710 --> 00:47:26,982 In yeast, it's brutal. 1075 00:47:26,982 --> 00:47:27,690 I mean, it works. 1076 00:47:27,690 --> 00:47:30,630 But it's the conditions are like it's a witch's brew. 1077 00:47:30,630 --> 00:47:34,680 Anyhow, so then you get the antibody in. 1078 00:47:34,680 --> 00:47:36,210 And that's what you're looking at. 1079 00:47:36,210 --> 00:47:37,630 And if you look in the-- 1080 00:47:37,630 --> 00:47:38,130 I have. 1081 00:47:38,130 --> 00:47:39,463 We're not going to get that far. 1082 00:47:39,463 --> 00:47:41,330 But I have pictures of-- 1083 00:47:41,330 --> 00:47:44,550 So when they compared the transient transfection 1084 00:47:44,550 --> 00:47:48,090 with the endogenous levels, that might give them 1085 00:47:48,090 --> 00:47:52,260 some feeling for what levels, the levels of expression 1086 00:47:52,260 --> 00:47:52,950 actually are. 1087 00:47:52,950 --> 00:47:56,670 And of course, the way that people really 1088 00:47:56,670 --> 00:48:01,200 want to attach things is using small things, 1089 00:48:01,200 --> 00:48:03,900 whatever these lists of dyes are that we have. 1090 00:48:03,900 --> 00:48:06,690 And what are the methods that you guys have learned about 1091 00:48:06,690 --> 00:48:09,330 to attach these fluorophores. 1092 00:48:09,330 --> 00:48:12,360 So instead of using a genetic fusion, which is probably. 1093 00:48:12,360 --> 00:48:14,940 That's a really good way, except the protein, 1094 00:48:14,940 --> 00:48:17,040 the green fluorescent protein is big. 1095 00:48:17,040 --> 00:48:19,440 Green fluorescent protein is also a dimer. 1096 00:48:19,440 --> 00:48:21,510 So people have spent a lot of time 1097 00:48:21,510 --> 00:48:24,390 engineering green fluorescent protein to be a monomer. 1098 00:48:24,390 --> 00:48:27,240 So the ones you buy commercially now are all monomers. 1099 00:48:27,240 --> 00:48:30,630 That would add complexity to everything on top of this. 1100 00:48:30,630 --> 00:48:33,000 How would you attach some of these things? 1101 00:48:33,000 --> 00:48:36,684 So we know what the structures of these things are. 1102 00:48:36,684 --> 00:48:38,390 AUDIENCE: You can do like a halo tag. 1103 00:48:38,390 --> 00:48:40,140 JOANNE STUBBE: So you could do a halo tag. 1104 00:48:40,140 --> 00:48:41,580 Have you talked-- we haven't talked about that. 1105 00:48:41,580 --> 00:48:42,780 So give me another method. 1106 00:48:42,780 --> 00:48:45,228 Give me a method we've talked about. 1107 00:48:45,228 --> 00:48:48,550 AUDIENCE: A handle, [INAUDIBLE] handle to attach? 1108 00:48:48,550 --> 00:48:52,407 JOANNE STUBBE: Yeah, but how would you do that? 1109 00:48:52,407 --> 00:48:53,740 How do you attach these handles? 1110 00:48:53,740 --> 00:48:55,130 You want to attach a fluorophore. 1111 00:48:55,130 --> 00:48:57,400 OK, so it turns out that all of these things 1112 00:48:57,400 --> 00:48:58,720 here, which you can't see. 1113 00:48:58,720 --> 00:49:04,240 But these little aromatic things have been synthesized. 1114 00:49:04,240 --> 00:49:05,470 So click it on. 1115 00:49:05,470 --> 00:49:06,970 If they could have a settling there. 1116 00:49:06,970 --> 00:49:09,482 But then it needs to be clicked to something. 1117 00:49:09,482 --> 00:49:11,210 AUDIENCE: [INAUDIBLE]. 1118 00:49:11,210 --> 00:49:12,850 JOANNE STUBBE: So you can't just. 1119 00:49:12,850 --> 00:49:13,890 So how do you click it? 1120 00:49:13,890 --> 00:49:16,280 AUDIENCE: [INAUDIBLE]. 1121 00:49:16,280 --> 00:49:19,630 JOANNE STUBBE: So but is that easy to do inside the cell? 1122 00:49:19,630 --> 00:49:20,130 No. 1123 00:49:20,130 --> 00:49:22,110 And in mammalian cells, it's impossible. 1124 00:49:22,110 --> 00:49:27,370 OK, so you can't use unnatural amino acids inside the cell. 1125 00:49:27,370 --> 00:49:30,890 The technology is not there at this stage. 1126 00:49:30,890 --> 00:49:33,917 So the question of how you attach this. 1127 00:49:33,917 --> 00:49:34,750 You could make your. 1128 00:49:34,750 --> 00:49:37,422 If you could make your protein outside the cell. 1129 00:49:37,422 --> 00:49:38,630 You might be able to do that. 1130 00:49:38,630 --> 00:49:40,838 But then you have the problem of getting your protein 1131 00:49:40,838 --> 00:49:41,810 inside the cell. 1132 00:49:41,810 --> 00:49:46,050 So getting a probe that's fluorescently, 1133 00:49:46,050 --> 00:49:50,960 you're labeling the protein of interest is not easy. 1134 00:49:50,960 --> 00:49:55,650 And Alice Ting's lab, again, has spent a lot of time, 1135 00:49:55,650 --> 00:49:57,220 not that successfully. 1136 00:49:57,220 --> 00:50:00,220 But using ligases that you can then 1137 00:50:00,220 --> 00:50:02,500 incorporate into the cells that can then 1138 00:50:02,500 --> 00:50:05,140 react with things you put onto your protein 1139 00:50:05,140 --> 00:50:06,860 to attach fluorophores. 1140 00:50:06,860 --> 00:50:08,860 But this is an area that's really important, 1141 00:50:08,860 --> 00:50:12,880 because in my opinion, looking at regulation inside the cell, 1142 00:50:12,880 --> 00:50:14,280 we don't really want to perturb. 1143 00:50:14,280 --> 00:50:16,520 We don't want to be at very high levels. 1144 00:50:16,520 --> 00:50:18,250 And we want to be able to see something 1145 00:50:18,250 --> 00:50:20,150 to understand regulation. 1146 00:50:20,150 --> 00:50:22,180 So I think. 1147 00:50:22,180 --> 00:50:25,450 So anyhow, the issue is that we want 1148 00:50:25,450 --> 00:50:27,830 to be as small as possible. 1149 00:50:27,830 --> 00:50:29,500 We don't want to be Brad's lab. 1150 00:50:29,500 --> 00:50:30,460 What is Brad's lab? 1151 00:50:30,460 --> 00:50:34,970 Does he use these nanobodies that are antibodies? 1152 00:50:34,970 --> 00:50:37,030 AUDIENCE: Like an [INAUDIBLE]? 1153 00:50:37,030 --> 00:50:38,740 JOANNE STUBBE: No. 1154 00:50:38,740 --> 00:50:39,700 They have all these. 1155 00:50:39,700 --> 00:50:41,980 They have things called the nanobodies now. 1156 00:50:41,980 --> 00:50:44,140 So I think they are like the little guys 1157 00:50:44,140 --> 00:50:46,760 you make on your solid phase peptide synthesizer. 1158 00:50:46,760 --> 00:50:48,700 But they are specific. 1159 00:50:48,700 --> 00:50:51,102 They specifically bind to proteins. 1160 00:50:51,102 --> 00:50:52,810 So there are only five examples that I've 1161 00:50:52,810 --> 00:50:53,530 seen in the literature. 1162 00:50:53,530 --> 00:50:54,700 So they act like antibodies. 1163 00:50:54,700 --> 00:50:56,190 But they're-- huh? 1164 00:50:56,190 --> 00:50:58,336 AUDIENCE: Like [INAUDIBLE],, like little-- 1165 00:50:58,336 --> 00:51:01,390 JOANNE STUBBE: They're little tiny proteins that are maybe. 1166 00:51:01,390 --> 00:51:02,080 I don't know. 1167 00:51:02,080 --> 00:51:06,250 50 amino acids that somehow, some guy at the University 1168 00:51:06,250 --> 00:51:08,290 of Chicago-- not Kent-- 1169 00:51:08,290 --> 00:51:09,848 developed these things. 1170 00:51:09,848 --> 00:51:10,765 And they specifically. 1171 00:51:10,765 --> 00:51:12,100 They act like an antibody. 1172 00:51:12,100 --> 00:51:15,570 They can specifically interact with a protein of interest. 1173 00:51:15,570 --> 00:51:18,160 And then you attach a green fluorescent protein onto it. 1174 00:51:18,160 --> 00:51:21,390 So again, what you have something smaller. 1175 00:51:21,390 --> 00:51:23,630 So because with these antibodies. 1176 00:51:23,630 --> 00:51:25,390 What you see is the non-specific, right? 1177 00:51:25,390 --> 00:51:27,075 I mean, we've seen that. 1178 00:51:27,075 --> 00:51:28,450 And with fluorescence, that means 1179 00:51:28,450 --> 00:51:32,330 you have fluorescence background in everything you do. 1180 00:51:32,330 --> 00:51:34,270 So anyhow, I think we're not that. 1181 00:51:34,270 --> 00:51:38,550 So that's just you're using fluorescence microscopy. 1182 00:51:38,550 --> 00:51:40,260 This tells you why you're interested 1183 00:51:40,260 --> 00:51:42,180 in fluorescence microscopy. 1184 00:51:42,180 --> 00:51:43,370 And we'll just close here. 1185 00:51:43,370 --> 00:51:47,320 And we're going to come back and talk about this in class. 1186 00:51:47,320 --> 00:51:50,040 But this is sort of the example of the data 1187 00:51:50,040 --> 00:51:51,310 that you need to think about. 1188 00:51:51,310 --> 00:51:54,120 So what we hear is in the presence of purines, 1189 00:51:54,120 --> 00:51:56,550 you don't see any of these little dots. 1190 00:51:56,550 --> 00:51:58,590 You remove the purines. 1191 00:51:58,590 --> 00:52:02,040 OK, so this is not so easy either, because the way 1192 00:52:02,040 --> 00:52:05,670 we grow cells, we don't have defined media, right? 1193 00:52:05,670 --> 00:52:06,660 I mean we're using. 1194 00:52:06,660 --> 00:52:09,180 I don't know what you guys use now, but fetal calf serum 1195 00:52:09,180 --> 00:52:10,877 or something. 1196 00:52:10,877 --> 00:52:12,960 It's got all this stuff in it that we don't really 1197 00:52:12,960 --> 00:52:13,830 know what it is. 1198 00:52:13,830 --> 00:52:16,390 We don't use defined media. 1199 00:52:16,390 --> 00:52:19,462 And apparently, when they-- the Marcotte paper-- 1200 00:52:19,462 --> 00:52:21,420 when they were describing this, said it was not 1201 00:52:21,420 --> 00:52:23,730 so easy to remove the purines. 1202 00:52:23,730 --> 00:52:26,640 And the method they used to remove the purines also 1203 00:52:26,640 --> 00:52:28,890 removed other stuff, OK. 1204 00:52:28,890 --> 00:52:30,480 So you're stressing the cell. 1205 00:52:30,480 --> 00:52:32,110 That was the take home message. 1206 00:52:32,110 --> 00:52:35,370 So under those conditions, you see something different. 1207 00:52:35,370 --> 00:52:37,860 OK, and so then they did another experiment, 1208 00:52:37,860 --> 00:52:40,500 because they were worried about levels. 1209 00:52:40,500 --> 00:52:42,870 Here, they are they have an antibody 1210 00:52:42,870 --> 00:52:44,780 to the trifuctional protein. 1211 00:52:44,780 --> 00:52:48,690 And so this is what they see under low purine conditions. 1212 00:52:48,690 --> 00:52:50,700 Does this look like this? 1213 00:52:50,700 --> 00:52:51,270 I don't know. 1214 00:52:51,270 --> 00:52:54,570 So you can't tell by looking at one picture. 1215 00:52:54,570 --> 00:52:57,750 OK, so you've got to do statistical analysis of all 1216 00:52:57,750 --> 00:52:58,300 these things. 1217 00:52:58,300 --> 00:53:01,320 So I think this sort of-- we'll come back and talk 1218 00:53:01,320 --> 00:53:04,090 about this in class. 1219 00:53:04,090 --> 00:53:06,615 But I think this is the first example where people 1220 00:53:06,615 --> 00:53:07,740 are trying to look at this. 1221 00:53:07,740 --> 00:53:09,430 The data is interesting. 1222 00:53:09,430 --> 00:53:12,180 But we've already raised issues of what some of the problems 1223 00:53:12,180 --> 00:53:12,680 are. 1224 00:53:12,680 --> 00:53:15,530 And hopefully, you can think about more of the problems.