1 00:00:05,080 --> 00:00:10,760 >>WELCOME TO TODAY'S 2 00:00:10,760 --> 00:00:12,000 NIH NEUROSCIENCE SEMINAR SERIES. 3 00:00:12,000 --> 00:00:14,520 IT'S A REAL PLEASURE--FOR THOSE 4 00:00:14,520 --> 00:00:17,640 WHO DON'T KNOW ME I AM 5 00:00:17,640 --> 00:00:19,520 [INDISCERNIBLE] I'M IN THE 6 00:00:19,520 --> 00:00:22,360 INTRAMURAL OF NICHD AND IT'S A 7 00:00:22,360 --> 00:00:25,120 REAL PLEASURE TO WELCOME AND 8 00:00:25,120 --> 00:00:29,160 INTRODUCE TODAY'S SPEAKER 9 00:00:29,160 --> 00:00:31,240 VASANTHI, JAYARAMAN. 10 00:00:31,240 --> 00:00:33,480 VASANTHI, GREW UP IN INDIA 11 00:00:33,480 --> 00:00:34,160 SPECIFICALLY IN [INDISCERNIBLE]. 12 00:00:34,160 --> 00:00:37,600 HE WENT TO HIS NATIVE 13 00:00:37,600 --> 00:00:38,040 [INDISCERNIBLE] FOR 14 00:00:38,040 --> 00:00:39,040 UNDERGRADUATE DEGREE AND THEN 15 00:00:39,040 --> 00:00:42,760 DID A MASTERS AT THE INDIAN 16 00:00:42,760 --> 00:00:43,920 INSTITUTE OF TECHNOLOGY IN 17 00:00:43,920 --> 00:00:45,560 [INDISCERNIBLE] WHERE I BELIEVE 18 00:00:45,560 --> 00:00:47,880 SHE WORKED WITH [INDISCERNIBLE] 19 00:00:47,880 --> 00:00:52,920 WHO'S A VERY PREEMINENT PHYSICAL 20 00:00:52,920 --> 00:00:55,120 CHEMIST BACK THERE IN INDIA AND 21 00:00:55,120 --> 00:00:56,680 THAT GOT HER EXCITED ABOUT 22 00:00:56,680 --> 00:00:59,520 CHEMISTRY AND THEN SHE WAS A 23 00:00:59,520 --> 00:01:02,320 GRADUATE STUDENT WITH TOM SPIRO 24 00:01:02,320 --> 00:01:04,720 AT THE CHEMISTRY DEPARTMENT AT 25 00:01:04,720 --> 00:01:09,440 PRINCETON UNIVERSITY. 26 00:01:09,440 --> 00:01:14,280 THERE VASANTHI, DID 27 00:01:14,280 --> 00:01:15,880 [INDISCERNIBLE] SPECROSCOPY TO 28 00:01:15,880 --> 00:01:16,760 UNDERSTAND HEMOGLOBIN AND SHE 29 00:01:16,760 --> 00:01:19,640 FOLLOWED THIS UP WITH A POST 30 00:01:19,640 --> 00:01:22,040 DOCTORAL FELLOWSHIP WHEN SHE WAS 31 00:01:22,040 --> 00:01:24,800 A POST DOCTORAL FELLOW IN GEORGE 32 00:01:24,800 --> 00:01:28,680 HESS'S LAB AT CORNELL UNIVERSITY 33 00:01:28,680 --> 00:01:30,480 WHERE SHE WAS--THAT WAS SORT OF 34 00:01:30,480 --> 00:01:31,920 HER INTRODUCTION TO THE WORLD OF 35 00:01:31,920 --> 00:01:33,480 PHYSIOLOGY AND ION CHANNELS. 36 00:01:33,480 --> 00:01:37,280 SO WHEN SHE STARTED, I THINK 37 00:01:37,280 --> 00:01:41,480 BACK IN 1997, IN THE CHEMISTRY 38 00:01:41,480 --> 00:01:42,280 DEPARTMENT AT [INDISCERNIBLE] 39 00:01:42,280 --> 00:01:46,120 UNIVERSITY SHE BROUGHT THE 2 40 00:01:46,120 --> 00:01:47,880 EXPERTISE TOGETHER COMBINING 41 00:01:47,880 --> 00:01:49,120 SPECROSCOPY TO STUDY ION CHANEMS 42 00:01:49,120 --> 00:01:53,080 AND THAT IS WHAT HER LAB HAS 43 00:01:53,080 --> 00:01:56,600 FOCUSED ON SINCE THEN. 44 00:01:56,600 --> 00:02:01,600 SHE HAS MOVED FROM WISCONSIN TO 45 00:02:01,600 --> 00:02:03,360 TD DEPARTMENT OF INTEGRATIVE 46 00:02:03,360 --> 00:02:05,280 BIOLOGY AT THE UNIVERSITY OF 47 00:02:05,280 --> 00:02:10,120 TEXAS IN 2002 AND HAS STAYED IN 48 00:02:10,120 --> 00:02:10,680 TEXAS SINCE THEN. 49 00:02:10,680 --> 00:02:13,880 AND NOW SHE'S A PROFESSOR IN THE 50 00:02:13,880 --> 00:02:15,240 DEPARTMENT OF CHEMISTRY AND 51 00:02:15,240 --> 00:02:16,400 MOLECULAR BIOLOGY AT THE 52 00:02:16,400 --> 00:02:17,920 UNIVERSITY OF TEXAS HEALTH 53 00:02:17,920 --> 00:02:21,000 CENTER, SHE'S GOTTEN NUMEROUS 54 00:02:21,000 --> 00:02:43,680 AWARDS AND SHE'S REALLY A 55 00:02:43,680 --> 00:02:45,880 SUCCESSFUL--HAS MOST RECENTLY 56 00:02:45,880 --> 00:02:48,120 BEEN APPOINTED AS CHIEF OF I 57 00:02:48,120 --> 00:02:49,240 BIOPHYSICAL JOURNAL SO WITH 58 00:02:49,240 --> 00:02:51,200 THAT, THANK YOU VERY MUCH FOR 59 00:02:51,200 --> 00:02:52,040 ACCEPTING OUR INVITATION AND I 60 00:02:52,040 --> 00:02:53,000 LOOK FORWARD TO YOUR TALK. 61 00:02:53,000 --> 00:03:00,640 WELCOME TO THE NIH. 62 00:03:00,640 --> 00:03:05,360 >> THANK YOU FOR THIS KIND 63 00:03:05,360 --> 00:03:06,880 INTRODUCTION, YOU CAN HEAR ME 64 00:03:06,880 --> 00:03:07,120 RIGHT? 65 00:03:07,120 --> 00:03:08,760 I ALSO WANTED TO JUST SAY THIS, 66 00:03:08,760 --> 00:03:11,240 WHEN HE SORT OF BROUGHT ALL THIS 67 00:03:11,240 --> 00:03:13,480 TOGETHER AND SAID, I BROUGHT IN 68 00:03:13,480 --> 00:03:15,880 THE SPECROSCOPY AND THE 69 00:03:15,880 --> 00:03:17,480 ELECTROPHYSIOLOGY TOGETHER, I DO 70 00:03:17,480 --> 00:03:21,480 WANT TO GIVE A SHOUT OUT AND SAY 71 00:03:21,480 --> 00:03:23,160 MARK MAYER IS THE 1 WHO TOLD ME, 72 00:03:23,160 --> 00:03:26,000 I DON'T THINK YOU CAN JUST DO 73 00:03:26,000 --> 00:03:26,960 JUST PHYSIOLOGY, THERE'S LOTS OF 74 00:03:26,960 --> 00:03:29,400 PEOPLE OUT THERE WHO ARE DOING 75 00:03:29,400 --> 00:03:30,680 THAT, BRING IN YOUR EXPERTISE IF 76 00:03:30,680 --> 00:03:32,480 YOU WANT TO DO SOMETHING USEFUL 77 00:03:32,480 --> 00:03:32,840 IN THIS FIELD. 78 00:03:32,840 --> 00:03:33,000 AND SO HERE I AM DOING COMBINING 79 00:03:33,000 --> 00:03:43,280 THE TWO 80 00:03:43,280 --> 00:03:44,560 RECEPTORS AND 81 00:03:44,560 --> 00:03:47,120 WORK WE'VE DONE ON SINGLE 82 00:03:47,120 --> 00:03:48,840 MOLECULE AND MORE RECENTLY GONE 83 00:03:48,840 --> 00:04:08,360 ON TO COMPLEXES IN THE SYNAPSES. 84 00:04:08,360 --> 00:04:10,160 --MOST OF THE WORK DONE BY DREW 85 00:04:10,160 --> 00:04:14,160 AND RYAN AND NITTY AND MABINA 86 00:04:14,160 --> 00:04:16,920 DID THE MD SIMULATIONS AND SHE'S 87 00:04:16,920 --> 00:04:32,080 ALSO DONE WORK ON RECEPTORS. 88 00:04:32,080 --> 00:04:35,040 SO THEY BIND GLUTAMATE AND THEY 89 00:04:35,040 --> 00:04:36,160 OPEN CHANNELS WHICH ARE CAT ION 90 00:04:36,160 --> 00:04:40,040 SPVENG AND THAT TRIGGERS THE 91 00:04:40,040 --> 00:04:42,960 ELECTRICAL SIGNALING AND WE USE 92 00:04:42,960 --> 00:04:44,360 A WIDE RANGE OF TECHNIQUES, 93 00:04:44,360 --> 00:04:48,280 EXPECTATIONS LECT ROUGH ATOM 94 00:04:48,280 --> 00:04:49,560 PHYSIOLOGY FUNCTIONAL STUDIES 95 00:04:49,560 --> 00:04:53,000 EXPW DOING FLOURESCENCE WE USE 96 00:04:53,000 --> 00:04:54,320 MOLECULAR BIOLOGY TO USE THE 97 00:04:54,320 --> 00:04:56,840 OTHER FORCE AND SO ON. 98 00:04:56,840 --> 00:05:00,160 WE DO A BIT OF CHEMISTRY SO 99 00:05:00,160 --> 00:05:01,480 WE'VE DONE DAIJ SYNTHESIS SO I 100 00:05:01,480 --> 00:05:03,120 WON'T TALK ABOUT IT BUT PUT IT 101 00:05:03,120 --> 00:05:05,560 OUT THERE IN CASE ANYBODY'S 102 00:05:05,560 --> 00:05:09,320 INTERESTED IN THAT. 103 00:05:09,320 --> 00:05:10,560 MOST OF OUR WORK, SO WE HAVE 104 00:05:10,560 --> 00:05:13,480 DONE ALL THE WAY FROM 105 00:05:13,480 --> 00:05:14,880 VIBRATIONAL FLUORESCENCE AND 106 00:05:14,880 --> 00:05:16,160 MAINLY SINGLE MOLECULE 107 00:05:16,160 --> 00:05:19,000 FLUORESCENCE AND LIFETIME 108 00:05:19,000 --> 00:05:19,360 MEASUREMENTS. 109 00:05:19,360 --> 00:05:22,480 AND WE COMPLEMENT THESE WITH MD 110 00:05:22,480 --> 00:05:25,520 SIMULATIONS WITH ALEX'S LAB AT 111 00:05:25,520 --> 00:05:25,680 UT. 112 00:05:25,680 --> 00:05:28,720 SO MOST OF WHAT I WILL BE 113 00:05:28,720 --> 00:05:30,960 TALKING ABOUT TODAY WILL FOCUS 114 00:05:30,960 --> 00:05:33,360 ON NMDA RECEPTORS AND THEN GO 115 00:05:33,360 --> 00:05:36,760 INTO DELTA OR MOST RECENT WORK 116 00:05:36,760 --> 00:05:37,360 ON DELTA RECEPTORS. 117 00:05:37,360 --> 00:05:39,560 SO A LITTLE BIT ABOUT MDA 118 00:05:39,560 --> 00:05:40,520 RECEPTORS, THE STRUCTURES ARE 119 00:05:40,520 --> 00:05:43,440 WELL KNOWN AND SO YOU HAVE--IT'S 120 00:05:43,440 --> 00:05:44,680 LIKE ALL THE GLUTAMATE 121 00:05:44,680 --> 00:05:47,320 RECEPTORS, IT IS A DIMER OF 122 00:05:47,320 --> 00:05:49,640 DIMERS, EXCEPT THE INTERESTING 123 00:05:49,640 --> 00:05:52,200 PART ABOUT NMDA RECEPTORS, HAVE 124 00:05:52,200 --> 00:05:54,280 YOU BOTH THE GLYSEEN BINDING 125 00:05:54,280 --> 00:06:04,160 DOMAIN AND YOU NEED BOTH GLYSEEN 126 00:06:04,160 --> 00:06:09,120 AND GLUTAMATE HAVE NEGATIVE 127 00:06:09,120 --> 00:06:09,480 COPRAATIVE. 128 00:06:09,480 --> 00:06:12,960 SO IN OTHER WORDS THEN IT IS 129 00:06:12,960 --> 00:06:13,160 LOW. 130 00:06:13,160 --> 00:06:17,160 IF GLUTAMATE IS BOND THEN 131 00:06:17,160 --> 00:06:18,920 GLIESINE IS LOW, SO WE WANT TO 132 00:06:18,920 --> 00:06:20,920 TEST THE UNDERLYING MECHANISM OF 133 00:06:20,920 --> 00:06:48,720 THAT ASPECT OF NMDA RECEPTORS. 134 00:06:48,720 --> 00:06:50,560 GLYSEEN IS FASTER VERSUS THE 135 00:06:50,560 --> 00:06:54,080 ABSENCE, AND THIS IN CASE WE ARE 136 00:06:54,080 --> 00:06:55,080 MEASURING DISASSOCIATION AND 137 00:06:55,080 --> 00:06:56,320 THEN IT'S SEEN HOW MUCH CURRENT 138 00:06:56,320 --> 00:06:58,120 AND LEFT BY ADDING GLUTAMATE. 139 00:06:58,120 --> 00:07:01,480 AND SAME AS THE CASE THE OTHER 140 00:07:01,480 --> 00:07:02,880 WAY AROUND WHERE GLUTAMATE 141 00:07:02,880 --> 00:07:05,080 ASSOCIATION IS FASTER IN THE 142 00:07:05,080 --> 00:07:07,720 PRESENCE OF GLYSEEN VERSUS IN 143 00:07:07,720 --> 00:07:11,960 THE ABSENCE OF GLYSEEN. 144 00:07:11,960 --> 00:07:13,960 SOPHISTICATED WHAT UTILIZES THIS 145 00:07:13,960 --> 00:07:16,280 SO FIRST THIS IS THE LIGAND 146 00:07:16,280 --> 00:07:18,480 BINDING DOMAIN AND IT'S SIMILAR 147 00:07:18,480 --> 00:07:20,720 HERE, THE GLUTAMATE BINDING 148 00:07:20,720 --> 00:07:21,840 DOMAIN, AND IF YOU CAN ZOOM IN, 149 00:07:21,840 --> 00:07:25,480 CAN YOU SEE THIS IS SORT OF THE 150 00:07:25,480 --> 00:07:27,160 CLEFT THAT IT BINDS AND IT'S 151 00:07:27,160 --> 00:07:28,760 KNOWN THAT WHENLET BI LAND 152 00:07:28,760 --> 00:07:31,880 BIEBDS THE CLEFT CLOSES SO IN 153 00:07:31,880 --> 00:07:33,600 ORDER TO MEASURE THE 154 00:07:33,600 --> 00:07:35,640 CONFIRMATIONAL CHANGE OF THE 155 00:07:35,640 --> 00:07:40,880 CLOSURE, THE USED FRONT AND WE 156 00:07:40,880 --> 00:07:41,840 INTRODUCED 2 CYSTINS IN 157 00:07:41,840 --> 00:07:43,480 [INDISCERNIBLE], SO WE REMOVE 158 00:07:43,480 --> 00:07:44,600 ALL THE CYSTINES IN THE EXTRA 159 00:07:44,600 --> 00:07:46,040 CELLULAR SIDE OF THE PROTEIN 160 00:07:46,040 --> 00:07:49,920 THAT ALLOWS US TO ABLE THE CELLS 161 00:07:49,920 --> 00:07:52,520 THAT CYSTINE REACTIVE FLORA FORS 162 00:07:52,520 --> 00:07:56,280 AND THE INTRODUCE THE CYSTINES 163 00:07:56,280 --> 00:07:56,720 IN THE SITES. 164 00:07:56,720 --> 00:07:58,560 Y SO YOU CAN MEASURE THE 165 00:07:58,560 --> 00:08:00,080 DISTANCE BETWEEN THESE 2-POINTS 166 00:08:00,080 --> 00:08:02,680 BY USING FRET, BECAUSE WHEN FRET 167 00:08:02,680 --> 00:08:03,920 WHEN YOU MEASURE THE AMOUNT OF 168 00:08:03,920 --> 00:08:06,600 LIGHT BEING DONATED FROM THE 169 00:08:06,600 --> 00:08:09,360 DONOR TO THE ACCEPTOR, YOU CAN 170 00:08:09,360 --> 00:08:10,680 SEE THE EFFICIENCIES DEPENDENTOT 171 00:08:10,680 --> 00:08:13,560 DISTANCE BETWEEN THE DONOR AND 172 00:08:13,560 --> 00:08:13,960 THE ACCEPTOR. 173 00:08:13,960 --> 00:08:15,440 ALL OF OUR MEASUREMENTS ARE DONE 174 00:08:15,440 --> 00:08:17,280 ON THE FULL LENGTH PROTEIN, SO 175 00:08:17,280 --> 00:08:19,560 YOU KNOW EVEN THOUGH I ZOOMED IN 176 00:08:19,560 --> 00:08:21,120 ON THE ISOLATED DOMAIN AND THE 177 00:08:21,120 --> 00:08:25,520 WAY WE DO IT IS WE EXPRESS THE 178 00:08:25,520 --> 00:08:25,960 PROTEIN? 179 00:08:25,960 --> 00:08:27,800 HEK CELLS, THE MODIFICATION IS 180 00:08:27,800 --> 00:08:29,440 IT'S A PROTEIN AND YOU HAVE 181 00:08:29,440 --> 00:08:32,120 THOSE IN THE SITE WHERE THE 182 00:08:32,120 --> 00:08:33,280 FLORA FLOR WAS INTRODUCED AND 183 00:08:33,280 --> 00:08:36,280 THE OTHER IS THIS WE HAVE A 184 00:08:36,280 --> 00:08:38,360 CANNED WHAT TAG AT C-TERMINUS AT 185 00:08:38,360 --> 00:08:48,280 THE END OF THE PROTEIN, AND 186 00:08:48,280 --> 00:08:51,520 EXPRESS THE PROTEIN AND WE DO AN 187 00:08:51,520 --> 00:09:02,960 NC2 PULL DOWN SO YOU CAN SEE SO 188 00:09:02,960 --> 00:09:07,760 THAT YOU CAN SEE THAT THE VENUE 189 00:09:07,760 --> 00:09:11,200 INTRODUCED THE CELL LYSAIT, YOU 190 00:09:11,200 --> 00:09:12,560 GET INSITU PULL DOWN OFF YOUR 191 00:09:12,560 --> 00:09:14,560 PROTEIN ON THE SLIDE AND THE 192 00:09:14,560 --> 00:09:15,920 MOLECULES ARE FAR APART BECAUSE 193 00:09:15,920 --> 00:09:19,880 WE ARE DILUTED THE BIOTIN ON THE 194 00:09:19,880 --> 00:09:21,560 SLIDE, AND SO YOU CAN SEE THAT 195 00:09:21,560 --> 00:09:23,360 YOU CAN ACTUALLY SEE INDIVIDUAL 196 00:09:23,360 --> 00:09:25,520 MOLECULES IN THE MICROSCOPE AND 197 00:09:25,520 --> 00:09:26,720 THEN YOU POINT TO 1 OF THESE 198 00:09:26,720 --> 00:09:29,800 MOLECULES AND YOU LOOK AT THE 199 00:09:29,800 --> 00:09:32,960 INTENSITY OF THAT MOLECULE IN 200 00:09:32,960 --> 00:09:36,200 BOTH THE DONOR AND ACCEPTOR 201 00:09:36,200 --> 00:09:37,560 FREQUENCIES OR WAVE LENGTHS SO 202 00:09:37,560 --> 00:09:39,680 THIS IS BASICALLY DONOR 203 00:09:39,680 --> 00:09:40,840 EXCITATION, BUT WHAT YOU ARE 204 00:09:40,840 --> 00:09:46,480 SEEING HERE IS THE ACCEPTAR 205 00:09:46,480 --> 00:09:48,560 EMISSION AND AND BELOW THAT IS 206 00:09:48,560 --> 00:09:50,600 THE DONOR EMISSION, AND AS CAN 207 00:09:50,600 --> 00:09:52,040 YOU AS YOU'RE INSIGHTING THE 208 00:09:52,040 --> 00:09:53,480 DONOR, THEY ARE GIVING THAT 209 00:09:53,480 --> 00:09:54,920 ENERGY TO THE ACCEPTOR AND THIS 210 00:09:54,920 --> 00:09:56,680 IS THE FRETTING REGION AND WE 211 00:09:56,680 --> 00:09:58,960 MEASURE 1 MOLECULE AT A TIME SO 212 00:09:58,960 --> 00:10:00,080 WE CAN ACTUALLY MEASURE THESE 213 00:10:00,080 --> 00:10:01,720 SPECIFIC DYNAMICS AND YOU CAN 214 00:10:01,720 --> 00:10:05,560 SEE THAT AT SOME POINT, THE 215 00:10:05,560 --> 00:10:07,000 ACCEPTOR BLEACHES AND YOU CAN 216 00:10:07,000 --> 00:10:09,400 SEE BECAUSE THE DONOR CAN NO 217 00:10:09,400 --> 00:10:11,480 LONGER GIVE ITS ENERGY TO THE 218 00:10:11,480 --> 00:10:14,520 ACCEPTOR, THEN THE DONOR COMES 219 00:10:14,520 --> 00:10:17,240 UP AND THIS CLEARLY SHOWINGS 220 00:10:17,240 --> 00:10:18,360 IT'S A SINGLE FLOR O BLEACHING 221 00:10:18,360 --> 00:10:20,680 STEP SO IT'S A SINGLE ACCEPTOR 222 00:10:20,680 --> 00:10:22,400 AND EVENTUALLY THE DONOR 223 00:10:22,400 --> 00:10:23,760 BLEACHES, SO THAT'S A SINGLE 224 00:10:23,760 --> 00:10:25,280 DONOR SO THE PROTEIN HAS A 225 00:10:25,280 --> 00:10:26,960 SINGLE DONOR AND ALSO YOU KNOW 226 00:10:26,960 --> 00:10:28,560 THAT THE ANTICORRELATION MEANS 227 00:10:28,560 --> 00:10:32,560 THAT YOU'RE FRETTING MOLECULES. 228 00:10:32,560 --> 00:10:32,760 OKAY? 229 00:10:32,760 --> 00:10:32,960 YEAH? 230 00:10:32,960 --> 00:10:34,400 >> [INDISCERNIBLE] 231 00:10:34,400 --> 00:10:39,960 >> THIS IS IN DETERGENT BUT WE 232 00:10:39,960 --> 00:10:40,440 ALSO DO SMA. 233 00:10:40,440 --> 00:10:42,960 >> AS FAR AS CONCERNS WE DON'T 234 00:10:42,960 --> 00:10:44,120 SEE ANY BIG DIFFERENCE WHETHER 235 00:10:44,120 --> 00:10:46,760 YOU DO THE SMA ACID POLYMER THAT 236 00:10:46,760 --> 00:10:51,240 YOU CAN USE TO MAKE NANO DISKS 237 00:10:51,240 --> 00:10:53,840 OFF THE PROTEIN AND AND WE FIND 238 00:10:53,840 --> 00:10:56,680 IT PUNCHES HOLES IN SIMPLISTIC 239 00:10:56,680 --> 00:10:57,840 TERMS OFFCELLS AND WE FIND THERE 240 00:10:57,840 --> 00:11:00,440 ARE SIMILAR IN THE EXTRA 241 00:11:00,440 --> 00:11:02,320 CELLULAR DOMAIN ABE AND WE SEE 242 00:11:02,320 --> 00:11:07,520 DIFFERENCES IN THE C-TERMINUS. 243 00:11:07,520 --> 00:11:09,200 EVERYTHING IS THERE, LIKE I SAID 244 00:11:09,200 --> 00:11:11,560 THE ONLY MODIFICATION WE DID WAS 245 00:11:11,560 --> 00:11:12,760 EXTRA CELLULAR THINGS REMOVED 246 00:11:12,760 --> 00:11:20,560 AND THAT'S A VERY CRITICAL 247 00:11:20,560 --> 00:11:20,800 POINT. 248 00:11:20,800 --> 00:11:22,360 WHAT YOU ARE SEEING HERE IS THIS 249 00:11:22,360 --> 00:11:23,520 REGION AND THIS PARTICULAR 250 00:11:23,520 --> 00:11:27,080 MOLECULE IS DO NOT DOING MUCH 251 00:11:27,080 --> 00:11:28,160 IT'S STAYING IN 1 STATE WHICH IS 252 00:11:28,160 --> 00:11:30,360 FINE AND THE GREEN IS THE 253 00:11:30,360 --> 00:11:32,400 OBSERVED AND THE BLACK IS THE 254 00:11:32,400 --> 00:11:33,840 D-NOISE DATA. 255 00:11:33,840 --> 00:11:35,000 SO REALLY ALL OF WHAT I WILL 256 00:11:35,000 --> 00:11:36,680 SHOW YOU IS BASED ON CHANGES 257 00:11:36,680 --> 00:11:39,120 THAT WE SEE IN THE GREEN BUT THE 258 00:11:39,120 --> 00:11:41,360 BLACK ALLOWS US TO GET BETTER, 259 00:11:41,360 --> 00:11:43,280 YOU KNOW IN TERMS OF FINDING OUT 260 00:11:43,280 --> 00:11:45,360 THE STATES, ET CETERA, SO THAT'S 261 00:11:45,360 --> 00:11:51,760 JUST THE DENOISING THAT WE DO. 262 00:11:51,760 --> 00:11:53,360 SO THIS IS THE HISTOGRAM FOR THE 263 00:11:53,360 --> 00:11:55,720 NERVE AND THIS IS THE HISTOGRAM, 264 00:11:55,720 --> 00:11:57,880 AND YOU CAN SEE IN THESE CASES 265 00:11:57,880 --> 00:11:59,560 IT'S REALLY ONLY 1 STATEMENT THE 266 00:11:59,560 --> 00:12:04,160 PROTEIN IS OCCUPYING. 267 00:12:04,160 --> 00:12:06,240 AND FROM THAT YOU CAN SEE WHAT 268 00:12:06,240 --> 00:12:08,160 WE CAN GET, BECAUSE WE HAVE ALL 269 00:12:08,160 --> 00:12:10,080 THE INTENSITIES, WE HAVE THE 270 00:12:10,080 --> 00:12:12,000 PRESENCE OF INTENSITY IN THE 271 00:12:12,000 --> 00:12:13,720 DONOR, IN IPT GREATER TENSITY IN 272 00:12:13,720 --> 00:12:15,720 THE ABSENCE OF THE DONOR AND SO 273 00:12:15,720 --> 00:12:19,040 SO ON SO WE CAN COUNT FROM THE 274 00:12:19,040 --> 00:12:19,280 THREAD. 275 00:12:19,280 --> 00:12:21,560 THE EXAMPLES I WILL SHOW YOU IS 276 00:12:21,560 --> 00:12:24,680 JUST THIS REGION, THE FRETTING 277 00:12:24,680 --> 00:12:25,520 REGION, NOT BEYOND THAT SO YOU 278 00:12:25,520 --> 00:12:27,960 CAN SEE THAT IN THE FRETTING 279 00:12:27,960 --> 00:12:30,600 REGION THIS PARTICULAR MOLECULE 280 00:12:30,600 --> 00:12:32,360 IS ACTUALLY SWITCHING BETWEEN 2 281 00:12:32,360 --> 00:12:33,840 DIFFERENT STATES, RIGHT? 282 00:12:33,840 --> 00:12:35,800 SO THIS IS THE ACCEPTOR, THIS IS 283 00:12:35,800 --> 00:12:39,240 THE DONOR AND THEN AT SOME 284 00:12:39,240 --> 00:12:40,760 POINT, THE THREAT BECOMES LESS 285 00:12:40,760 --> 00:12:42,960 SO THE DONOR GOES UP AND THE 286 00:12:42,960 --> 00:12:44,960 ACCEPTOR GOES DOWN AND 2 THREAT 287 00:12:44,960 --> 00:12:47,160 EFFICIENCIES AND YOU CAN SEE 288 00:12:47,160 --> 00:12:48,760 IT'S STAYED IF ARE A WHILE 289 00:12:48,760 --> 00:12:50,160 BEFORE IT SWITCHES INTO THE 290 00:12:50,160 --> 00:12:50,600 OTHER STATE. 291 00:12:50,600 --> 00:12:51,560 THE DIFFERENCE BETWEEN THIS 292 00:12:51,560 --> 00:12:52,520 MOLECULE AND THIS MOLECULE IS 293 00:12:52,520 --> 00:12:55,600 THAT EVEN THOUGH THIS IS SORT OF 294 00:12:55,600 --> 00:12:58,160 SIMILAR 2 STATES, YOU SEE THAT 295 00:12:58,160 --> 00:13:00,240 HERE THE MOLECULE IS ACTUALLY 296 00:13:00,240 --> 00:13:02,960 FLUCTUATING A LOT AND EVERY 297 00:13:02,960 --> 00:13:05,640 THRUCTUATION YOU GET, THERE IS 298 00:13:05,640 --> 00:13:08,680 ANTICORRELATION SAYING IT IS 299 00:13:08,680 --> 00:13:10,400 REALLY THREAT DIFFERENCES AND 300 00:13:10,400 --> 00:13:12,160 THIS MOLECULE IS FLUCTUATING 301 00:13:12,160 --> 00:13:12,720 BETWEEN THE 2 STATES. 302 00:13:12,720 --> 00:13:15,240 SO WHAT YOU GET FROM SINGLE 303 00:13:15,240 --> 00:13:16,560 MOLECULE THREAT IS NOT JUST THE 304 00:13:16,560 --> 00:13:19,640 THREAT DISTANCES BUT ALSO THE 305 00:13:19,640 --> 00:13:21,760 DYNAMICS OFF THE PROTEIN IN HOW 306 00:13:21,760 --> 00:13:23,640 IT SWITCHES BETWEEN THE STAKES, 307 00:13:23,640 --> 00:13:25,280 BUT THE DYNAMIC SYSTEM LIMITED 308 00:13:25,280 --> 00:13:27,240 IN THE MILLI SECOND TIME SCALE, 309 00:13:27,240 --> 00:13:29,920 IT'S NOT FASTER OR VERY LONG OR 310 00:13:29,920 --> 00:13:31,000 SLOWER DYNAMICS ARE ALSO GOING 311 00:13:31,000 --> 00:13:32,760 TO BE MISSED BECAUSE HAVE YOU 312 00:13:32,760 --> 00:13:33,160 PHOTO BLEACHING. 313 00:13:33,160 --> 00:13:36,840 SO IT'S SORT OF IN THE SWEET 314 00:13:36,840 --> 00:13:38,960 SPOT OF 5 MILLI SECONDS, 200 315 00:13:38,960 --> 00:13:42,280 MILLI SECONDS. 316 00:13:42,280 --> 00:13:42,520 OKAY? 317 00:13:42,520 --> 00:13:44,480 SO THIS IS WHAT RYAN FOUND WHEN 318 00:13:44,480 --> 00:13:47,040 HE DID THE ISOLATED LIGAND 319 00:13:47,040 --> 00:13:49,240 BINDING DOMAIN AND THE GRAY IS 320 00:13:49,240 --> 00:13:51,080 THE OBSERVED DATA AND THE BLUE 321 00:13:51,080 --> 00:13:52,160 IS JUST THE DENOISING TO SEE 322 00:13:52,160 --> 00:13:53,360 WHAT STATES ARE THERE, BUT 323 00:13:53,360 --> 00:13:56,160 PRETTY MUCH ALL OF OUR 324 00:13:56,160 --> 00:13:56,760 MEASUREMENTS, CAN YOU JUST 325 00:13:56,760 --> 00:13:58,960 OVERLOAD THE GRAY AND SEE THE 326 00:13:58,960 --> 00:14:00,120 DIFFERENCES, OKAY, EVEN THOUGH 327 00:14:00,120 --> 00:14:04,560 YOU'RE SEEING THE BLUE, BEING 328 00:14:04,560 --> 00:14:05,760 SHOWN HERE, IT'S EVEN WITH THE 329 00:14:05,760 --> 00:14:07,480 OBSERVED DATA YOU CAN SEE WHAT'S 330 00:14:07,480 --> 00:14:07,960 GOING ON. 331 00:14:07,960 --> 00:14:09,960 BUT BOTTOM LINE IS THIS THE APO 332 00:14:09,960 --> 00:14:13,000 STATE WHEN THERE IS NO LIGAND IN 333 00:14:13,000 --> 00:14:14,560 BOTH SIDES AND WHEN MOTIONING 334 00:14:14,560 --> 00:14:15,720 THE GLUTAMATE, THE GLUE MARIOUS 335 00:14:15,720 --> 00:14:17,360 SEEN BINDING SITED, YOU SEE IT'S 336 00:14:17,360 --> 00:14:20,120 AT A CERTAIN DISTANCE, AGAIN THE 337 00:14:20,120 --> 00:14:20,960 ABSOLUTE DISTANCE DOESN'T MEAN 338 00:14:20,960 --> 00:14:25,440 MUCH BECAUSE YOU'RE MEASURING 339 00:14:25,440 --> 00:14:26,880 BETWEEN FLORA FLORS BUT ALSO I 340 00:14:26,880 --> 00:14:28,360 WOULD LIKE YOU TO SEE THE 341 00:14:28,360 --> 00:14:29,600 TRANSITIONS I SHOWED YOU IN THE 342 00:14:29,600 --> 00:14:30,600 PREVIOUS SLIDE AND THIS SORT OF 343 00:14:30,600 --> 00:14:32,800 GIVES YOU THE NUMBER OF 344 00:14:32,800 --> 00:14:34,080 TRANSITIONS THAT ARE HAPPENING 345 00:14:34,080 --> 00:14:34,920 BETWEEN THE DIFFERENT STATES AND 346 00:14:34,920 --> 00:14:36,440 YOU CAN SEE THAT THERE'S A 347 00:14:36,440 --> 00:14:38,440 CERTAIN NUMBER OF TRANSITIONS 348 00:14:38,440 --> 00:14:40,080 THAT IS HAPPENING IN THEA, PO 349 00:14:40,080 --> 00:14:40,280 STATE. 350 00:14:40,280 --> 00:14:42,600 NOW WHEN YOU GO TO THE GLUE 351 00:14:42,600 --> 00:14:44,360 MARIOUS SEEN STATE, YES, THE 352 00:14:44,360 --> 00:14:46,400 CLEFT CLOSES AND THIS IS 353 00:14:46,400 --> 00:14:48,160 CLEARLY, IT'S KNOWN IN THE 354 00:14:48,160 --> 00:14:51,160 STRUCTURE SO IT'S NOT LIKE WE 355 00:14:51,160 --> 00:14:53,640 ARE SHOWING THAT THE CLEFT 356 00:14:53,640 --> 00:14:55,840 CLOSED, THIS HAS BEEN KNOWN FOR 357 00:14:55,840 --> 00:14:57,040 A WHILE, BUT WHAT IS INTERESTING 358 00:14:57,040 --> 00:14:58,240 IS THAT THE TRANSITIONS ARE VERY 359 00:14:58,240 --> 00:15:01,200 LESS AND THE PROTEIN IS VERY 360 00:15:01,200 --> 00:15:01,440 STABLE. 361 00:15:01,440 --> 00:15:02,760 THAT'S WHAT YOU EXPECT, RIGHT? 362 00:15:02,760 --> 00:15:03,960 YOU HAVE A GLUE MARIOUS SEEN 363 00:15:03,960 --> 00:15:07,360 BOUND AND THEN CLEARLY THE 364 00:15:07,360 --> 00:15:08,600 INTERACTION WILL STABILIZE THE 365 00:15:08,600 --> 00:15:10,360 CLEFT AND SO THEREFORE THE 366 00:15:10,360 --> 00:15:11,840 TRANSITION WILL BE REALIZED. 367 00:15:11,840 --> 00:15:13,960 IT'S NOT SURPRISING BUT AGAIN IT 368 00:15:13,960 --> 00:15:16,680 CONFIRMED WHAT WE WOULD THINK 369 00:15:16,680 --> 00:15:17,000 SHOULD HAPPEN. 370 00:15:17,000 --> 00:15:18,560 BUT WHAT IS INTERESTING IS WHEN 371 00:15:18,560 --> 00:15:21,080 YOU ADD GLUTAMATE TO THE OTHER 372 00:15:21,080 --> 00:15:24,760 SITE, WHAT YOU SEE IS GLYSEEN 373 00:15:24,760 --> 00:15:25,960 BINDING SITE, SLIGHTLY SHIFTS 374 00:15:25,960 --> 00:15:27,160 BETWEEN THESE STATES WHERE IT'S 375 00:15:27,160 --> 00:15:28,880 GOING MORE INTO THE OPEN STATE 376 00:15:28,880 --> 00:15:31,640 BUT IT'S A SLIGHT SHIFT BUT 377 00:15:31,640 --> 00:15:33,080 AGAIN YOU CAN START SEEING THAT 378 00:15:33,080 --> 00:15:35,800 THE TRANSITIONS ARE GETTING 379 00:15:35,800 --> 00:15:36,000 MORE. 380 00:15:36,000 --> 00:15:37,280 SO THEN THE DPLIEWT MATE IS 381 00:15:37,280 --> 00:15:39,040 BOUND, THE GLUE MARIOUS SEEN 382 00:15:39,040 --> 00:15:39,920 BINDING CLEFT IS STARTING TO 383 00:15:39,920 --> 00:15:41,840 OPEN UP AND THE TRANSITIONS, 384 00:15:41,840 --> 00:15:45,320 IT'S A BIT MORE UNSTABLE THERE, 385 00:15:45,320 --> 00:15:46,520 IT'S STARTING TO FORM, IT'S 386 00:15:46,520 --> 00:15:48,480 STARTING TO HAVE MORE OF THESE 387 00:15:48,480 --> 00:15:50,320 TRANSITIONS, BUT EVEN THAT IS 388 00:15:50,320 --> 00:15:55,560 PROBABLY NOT AS DRAMATIC AS WHAT 389 00:15:55,560 --> 00:15:56,520 YOU SEE HERE. 390 00:15:56,520 --> 00:16:00,840 YOU DON'T HAVE GLYSEEN IN THE 391 00:16:00,840 --> 00:16:02,160 GLYSEEN BINDING SITE, YOU JUST 392 00:16:02,160 --> 00:16:02,720 HAVE GLUTAMATE AND YOU SLEEP 393 00:16:02,720 --> 00:16:04,360 APNEA AND OBESITYY THAT REALLY 394 00:16:04,360 --> 00:16:05,680 THE LIGAND BINDING DOMAIN IS 395 00:16:05,680 --> 00:16:08,240 LOOKING AT A LOT OF DIFFERENT 396 00:16:08,240 --> 00:16:09,520 CONFIRMATIONAL STATES AND 397 00:16:09,520 --> 00:16:12,240 THERE'S A LOT OF DYNAMICS, SO IN 398 00:16:12,240 --> 00:16:13,480 OTHER WORDS, THIS GLUTAMATE 399 00:16:13,480 --> 00:16:14,480 BINDING HERE IS AFFECTING THE 400 00:16:14,480 --> 00:16:17,480 GLUE MARIOUS SEEN BINDING SITE 401 00:16:17,480 --> 00:16:19,200 ALOESTERICALLY AND THAT IS SORT 402 00:16:19,200 --> 00:16:22,080 OF GOING TO AFFECT THE RATE AT 403 00:16:22,080 --> 00:16:25,000 WHICH GLYSEEN BINDS TO THE 404 00:16:25,000 --> 00:16:25,240 PROTEIN. 405 00:16:25,240 --> 00:16:28,160 SO THE SAME THING IS TRUE IN THE 406 00:16:28,160 --> 00:16:28,880 GLUTAMATE BINDING SITE AND I 407 00:16:28,880 --> 00:16:30,640 WILL NOT BORE YOU CAN THE DETAIL 408 00:16:30,640 --> 00:16:33,160 EXCEPT TO SAY THAT AGAIN, THE 409 00:16:33,160 --> 00:16:37,160 APO STATE IS MORE OPEN, MORE 410 00:16:37,160 --> 00:16:38,960 DYNAMIC, GLUTAMATE ALONE IS 411 00:16:38,960 --> 00:16:40,200 FAVORING MORE CLOSE, LESS 412 00:16:40,200 --> 00:16:42,720 DYNAMIC BUT THEN YOU HAVE 413 00:16:42,720 --> 00:16:43,480 GLYSEEN IN THE SITE. 414 00:16:43,480 --> 00:16:45,680 YOU SEE THAT THE DYNAMICS IS 415 00:16:45,680 --> 00:16:50,640 REALLY GETTING VERY LARGE, OKAY? 416 00:16:50,640 --> 00:16:51,840 SO TO MAKE SURE THAT THIS IS 417 00:16:51,840 --> 00:16:56,520 WHAT WE ARE MEASURING IS A 418 00:16:56,520 --> 00:16:57,760 POSSIBILITY, WE NEEDED MB 419 00:16:57,760 --> 00:16:59,320 SIMULATION BUT WE ALSO WANTED TO 420 00:16:59,320 --> 00:17:01,360 SEE WHAT INTERFACE IS 421 00:17:01,360 --> 00:17:02,760 CONTRIBUTING TO THIS NEGATIVE 422 00:17:02,760 --> 00:17:03,160 COOPERATIVE DEAL. 423 00:17:03,160 --> 00:17:06,880 AND YOU WILL SEE THAT IN THE 424 00:17:06,880 --> 00:17:07,840 NMDA RECEPTORS THERE'S 2 425 00:17:07,840 --> 00:17:11,000 DIFFERENT INTERFACES THIS, IS 1 426 00:17:11,000 --> 00:17:12,120 INTERFACE WHICH IS BASICALLY 427 00:17:12,120 --> 00:17:13,480 THIS IS THE GLUE MARIOUS SEEN 428 00:17:13,480 --> 00:17:15,120 BINDING SITE AND THAT'S 429 00:17:15,120 --> 00:17:16,640 GLUTAMATE BINDING SITE AND THIS 430 00:17:16,640 --> 00:17:18,400 IS THE OTHER INTERFACE WHEN YOU 431 00:17:18,400 --> 00:17:20,000 TURN AT 90-DEGREES AND THAT'S 432 00:17:20,000 --> 00:17:22,240 AGAIN THE GLUE MARIOUS SEEN AND 433 00:17:22,240 --> 00:17:25,920 GLUTAMATE, AND GLUE MARIOUS SEEN 434 00:17:25,920 --> 00:17:26,440 AND GLUTAMATE. 435 00:17:26,440 --> 00:17:28,280 SO WHAT THEY DID WAS TOOK JUST 436 00:17:28,280 --> 00:17:30,600 THE INTERFACES AND SAID LET'S DO 437 00:17:30,600 --> 00:17:32,120 AN MD SIMULATION AND SEE HOW 438 00:17:32,120 --> 00:17:34,160 GLUE MARIOUS SEEN ALONE OR 439 00:17:34,160 --> 00:17:36,360 GLUTAMATE ALONE OR GLUTAMATE AND 440 00:17:36,360 --> 00:17:39,800 LYSINE AFFECTS THE CLEFT BETWEEN 441 00:17:39,800 --> 00:17:41,760 THESE AND IF IT CAN BE SORT OF 442 00:17:41,760 --> 00:17:42,920 CORRELATED TO THE SINGLE 443 00:17:42,920 --> 00:17:44,600 MOLECULE THREAT WE GOT ON THE 444 00:17:44,600 --> 00:17:50,720 FULL LENGTH RECEPTOR, OKAY? 445 00:17:50,720 --> 00:17:52,560 AND SHE SAW THAT MEASURING THE 446 00:17:52,560 --> 00:17:54,560 SAME SITES AS THE SINGLE 447 00:17:54,560 --> 00:17:56,280 MOLECULE FRET, SHE FOUND THAT 448 00:17:56,280 --> 00:18:00,480 BASICALLY THE TRENDS THAT WE ARE 449 00:18:00,480 --> 00:18:02,760 SEEING THEN YOU HAVE GLUTAMATE 450 00:18:02,760 --> 00:18:04,560 AND GLYSEEN AND BINDING DOMAIN 451 00:18:04,560 --> 00:18:06,520 IS STARTING TO OPEN THE APO 452 00:18:06,520 --> 00:18:08,760 STATE AND THE MOST UNSTABLE 453 00:18:08,760 --> 00:18:10,960 BEING GLUTAMATE WHEN YOU ARE 454 00:18:10,960 --> 00:18:17,520 MEASURING AT THE GLSEEN AND 455 00:18:17,520 --> 00:18:18,160 GLYCINE FOR THE GLUTAMATE. 456 00:18:18,160 --> 00:18:19,080 THESE MATCH VERY WELL WHAT WE 457 00:18:19,080 --> 00:18:23,760 SAW IN THE SINGLE MOLECULE FRET, 458 00:18:23,760 --> 00:18:25,600 WHEN SHE DID THE BACK-TO-BACK, 459 00:18:25,600 --> 00:18:26,800 THE TRANSFER NOT UNTILING THE 460 00:18:26,800 --> 00:18:29,160 SAME AS WHAT WE SAW IN A SINGLE 461 00:18:29,160 --> 00:18:30,920 MOLECULE AND IN FACT IN SOME 462 00:18:30,920 --> 00:18:34,480 CASES, IT WOULD SORT OF GO TO 463 00:18:34,480 --> 00:18:35,800 POSITIVE COOPERATIVITY BECAUSE 464 00:18:35,800 --> 00:18:37,760 OF THE TRENDS. 465 00:18:37,760 --> 00:18:39,960 SO, WE THINK THAT WILD THIS 466 00:18:39,960 --> 00:18:41,600 INTERFACE MAY BE, I'M NOT SAYING 467 00:18:41,600 --> 00:18:42,920 IT'S NOT IMPORTANT, ALL I'M 468 00:18:42,920 --> 00:18:46,440 SAYING IS IT'S NOT COOPERATED 469 00:18:46,440 --> 00:18:48,600 HF-IT'S NOT CONTRIBUTING AS MUCH 470 00:18:48,600 --> 00:18:55,760 TO THE NEGATIVE COOPERATIVE. 471 00:18:55,760 --> 00:18:57,920 SO HAVING ESTABLISHED--SORRY, I 472 00:18:57,920 --> 00:18:58,800 WILL GO BACK. 473 00:18:58,800 --> 00:18:59,960 HAVING ESTABLISHED THAT THIS 474 00:18:59,960 --> 00:19:04,520 INTERFACE MIGHT BE CRITICAL FOR 475 00:19:04,520 --> 00:19:06,360 THE TEST, WE DECIDED TO CROSS 476 00:19:06,360 --> 00:19:09,280 LINK THIS USING A NATURAL AMINO 477 00:19:09,280 --> 00:19:12,080 ACID AND SEE IF INDEED THIS IS 478 00:19:12,080 --> 00:19:14,160 CROSS AND SHOULD BE AFFECTING 479 00:19:14,160 --> 00:19:14,920 NEGATIVE COOPERATIVELY. 480 00:19:14,920 --> 00:19:17,800 SO WE INTRODUCE A NATURAL AMINO 481 00:19:17,800 --> 00:19:18,960 ACID AND BE CROSS LINKED AND AS 482 00:19:18,960 --> 00:19:20,520 YOU CAN SEE THE RISK, SOME 483 00:19:20,520 --> 00:19:22,600 FRACTION OF THE PROTEIN THAT'S 484 00:19:22,600 --> 00:19:23,880 CROSS LINKED, WE CAN SEE AN 485 00:19:23,880 --> 00:19:26,760 EFFECT IF THAT IS AN EFFECT AND 486 00:19:26,760 --> 00:19:29,720 AS EXPECTED, THE NEGATIVE 487 00:19:29,720 --> 00:19:30,560 COOPERATIVITY IS SIGNIFICANTLY 488 00:19:30,560 --> 00:19:31,960 REDUCED, SO IF YOU SEE THIS IS 489 00:19:31,960 --> 00:19:36,600 THE WILD-TYPE, THIS IS THE--THE 490 00:19:36,600 --> 00:19:38,320 GLUTAMATE SO THE RATIO TELLS YOU 491 00:19:38,320 --> 00:19:40,280 IF THERE IS NEGATIVE 492 00:19:40,280 --> 00:19:40,800 COOPERATIVITY. 493 00:19:40,800 --> 00:19:43,000 SO THIS HAS NEGATIVE 494 00:19:43,000 --> 00:19:43,360 COOPERATIVITY. 495 00:19:43,360 --> 00:19:45,400 AND IT'S VERY SITE SPECIFIC, SO 496 00:19:45,400 --> 00:19:48,080 THEN YOU HAVE THIS UNNATURAL 497 00:19:48,080 --> 00:19:52,640 AMINO ACID BEING INTRODUCED AT 498 00:19:52,640 --> 00:19:52,920 720. 499 00:19:52,920 --> 00:19:54,440 YOU SEE THAT IT DOESN'T HAVE AN 500 00:19:54,440 --> 00:19:55,640 EFFECT BECAUSE IT'S NOT RIGHT AT 501 00:19:55,640 --> 00:19:57,840 THE INTERFACE BUT WHEN YOU 502 00:19:57,840 --> 00:19:59,560 INTRODUCE IT AT 723, YOU SEE IT 503 00:19:59,560 --> 00:20:01,880 HAS AN EFFECT AND AGAIN, AT 712 504 00:20:01,880 --> 00:20:04,160 IT DOESN'T. 505 00:20:04,160 --> 00:20:05,560 SO THIS INTERFACE WHICH IS RIGHT 506 00:20:05,560 --> 00:20:07,560 AT THE INTERFACE WHEN IT'S CROSS 507 00:20:07,560 --> 00:20:09,280 LINKED ACROSS, IT DOES HAVE AN 508 00:20:09,280 --> 00:20:10,920 EFFECT ON NEGATIVE 509 00:20:10,920 --> 00:20:11,960 COOPERATIVITY, FURTHER SHOWING 510 00:20:11,960 --> 00:20:15,680 THAT THIS INTERFACE IS CRITICAL 511 00:20:15,680 --> 00:20:16,400 FOR NEGATIVE COOPERATIVE. 512 00:20:16,400 --> 00:20:19,160 BUT AGAIN, I DON'T WANT YOU TO 513 00:20:19,160 --> 00:20:22,280 LEAVE THINKING THAT IS THE ONLY 514 00:20:22,280 --> 00:20:24,760 INTERFACE THAT'S IMPORTANT FOR 515 00:20:24,760 --> 00:20:28,560 THE ALOESTERRIC CROSS TALK 516 00:20:28,560 --> 00:20:30,680 BETWEEN GLYCINE AND GLUTAMATE, 517 00:20:30,680 --> 00:20:32,600 WE DID ANALYSIS WHERE HE CAME TO 518 00:20:32,600 --> 00:20:34,040 US AND SDZ, HEY, LISTEN WE HAVE 519 00:20:34,040 --> 00:20:36,080 THIS MUTATION IN THIS PATIENT 520 00:20:36,080 --> 00:20:38,040 AND OUR ELECTROPHYSIOLOGICAL 521 00:20:38,040 --> 00:20:39,200 STUDIES IS SHOWING THAT EVEN 522 00:20:39,200 --> 00:20:42,720 THOUGH THE MUTATION IS IN THE 523 00:20:42,720 --> 00:20:45,960 GLYCINE BINDING DOMAIN, IT'S NOT 524 00:20:45,960 --> 00:20:47,440 AFFECTING THE GLYCINE IN TERMS 525 00:20:47,440 --> 00:20:49,440 OF FUNCTION BUT IT IS AFFECTING 526 00:20:49,440 --> 00:20:51,040 GLUTAMATE BINDING IN TERMS OF 527 00:20:51,040 --> 00:20:53,360 FUNCTION AND THAT GLUTAMATE 528 00:20:53,360 --> 00:20:55,560 BINDING IS MUCH LESS EFFICIENT. 529 00:20:55,560 --> 00:20:56,960 SO AGAIN [INDISCERNIBLE] DID MD 530 00:20:56,960 --> 00:20:59,360 SIMULATIONINGS TO SEE HOW IT'S 531 00:20:59,360 --> 00:21:00,320 AFFECTING THIS MUTATION IS 532 00:21:00,320 --> 00:21:02,280 AFFECTING THE PROTEIN AND IT'S 533 00:21:02,280 --> 00:21:06,720 ACTUALLY AT THIS BACK-TO-BACK 534 00:21:06,720 --> 00:21:07,320 INTERFACE. 535 00:21:07,320 --> 00:21:10,240 IT'S A PROLEAN THAT'S BEEN 536 00:21:10,240 --> 00:21:12,320 MUTATED TO HISTIDINE, IT 537 00:21:12,320 --> 00:21:14,040 ACTUALLY FORMS A HYDROGEN 1 538 00:21:14,040 --> 00:21:15,320 ACROSS, THIS IS THE GLUE MARIOUS 539 00:21:15,320 --> 00:21:16,760 SEEN BINDING SITE AND THIS IS 540 00:21:16,760 --> 00:21:19,480 THE DPLIEWT MATE BINDING SITE. 541 00:21:19,480 --> 00:21:21,160 IT FORMS THIS HYDROGEN BINDING 542 00:21:21,160 --> 00:21:23,320 ACROSS AND KIND OF PROVES THE 543 00:21:23,320 --> 00:21:25,280 LOWER PART OF THE GLUTAMATE 544 00:21:25,280 --> 00:21:28,000 BINDING DOMAIN, SUCH THAT IT NOW 545 00:21:28,000 --> 00:21:29,960 SEEMS TO POP OPEN MORE THEREFORE 546 00:21:29,960 --> 00:21:33,560 GLUTAMATE IS NOT ABLE TO BIND AS 547 00:21:33,560 --> 00:21:34,160 EFFICIENTLY. 548 00:21:34,160 --> 00:21:36,600 SO IT'S IN THOSE TO BACK 549 00:21:36,600 --> 00:21:37,560 INTERFACE AND IT'S PULLING THIS 550 00:21:37,560 --> 00:21:39,680 THING MORE OPEN SO THAT NOT NOW 551 00:21:39,680 --> 00:21:40,840 IT'S NOT BINDING ASENTIOUS 552 00:21:40,840 --> 00:21:41,120 FICIENTLY. 553 00:21:41,120 --> 00:21:42,960 SO OF COURSE THIS IS MD 554 00:21:42,960 --> 00:21:44,960 SIMULATIONS, WE WANT TO VERIFY 555 00:21:44,960 --> 00:21:45,720 IT WITH SINGLE MOLECULE THREAT 556 00:21:45,720 --> 00:21:51,120 AND I WILL JUST SHOW YOU 557 00:21:51,120 --> 00:21:53,160 THE--AGAIN THE SAME LOOKING AT 558 00:21:53,160 --> 00:21:54,480 GLUTAMATE ACROSS AND SEEING HOW 559 00:21:54,480 --> 00:21:57,760 THE MOTIONS ARE, SO I WILL JUST 560 00:21:57,760 --> 00:22:03,400 SHOW YOU THE CATONNATED FRET 561 00:22:03,400 --> 00:22:05,080 STATES, YOU CAN SEE ALL THE FRET 562 00:22:05,080 --> 00:22:06,120 STATES THE PROTEIN IS EXLORING 563 00:22:06,120 --> 00:22:07,560 AND THIS IS THE WILD-TYPE AND 564 00:22:07,560 --> 00:22:09,480 THIS IS WHAT I SHOWED YOU 565 00:22:09,480 --> 00:22:10,320 BEFORE, THERE'S NOT MUCH MOTION 566 00:22:10,320 --> 00:22:11,880 YOU SEE IN THE WILD-TYPE BUT IN 567 00:22:11,880 --> 00:22:14,000 THE MUTANT YOU CAN SEE THAT IT'S 568 00:22:14,000 --> 00:22:16,600 FLUCTUATING A LOT BETWEEN THE 569 00:22:16,600 --> 00:22:17,040 STATES. 570 00:22:17,040 --> 00:22:19,120 AND IT'S GOING FROM A HIGH FRET 571 00:22:19,120 --> 00:22:22,040 STATE TO A MUCH LOWER FRET STATE 572 00:22:22,040 --> 00:22:23,480 THEREFORE THE CLEFT IS OPENING 573 00:22:23,480 --> 00:22:23,920 MORE. 574 00:22:23,920 --> 00:22:27,000 SO THIS SORT OF SHOWS YOU WHERE 575 00:22:27,000 --> 00:22:31,720 YOU GO FROM MUTATION FUNCTION TO 576 00:22:31,720 --> 00:22:32,880 MOLECULAR DYNAMIC SIMULATION TO 577 00:22:32,880 --> 00:22:34,920 SINGLE MOLECULE SHOW HOW THE 578 00:22:34,920 --> 00:22:36,640 DYNAMICS IS INVOLVED IN 579 00:22:36,640 --> 00:22:43,320 DICTATING FUNCTION. 580 00:22:43,320 --> 00:22:43,520 OKAY? 581 00:22:43,520 --> 00:22:46,960 SO JUST TO SHOW YOU A LITTLE BIT 582 00:22:46,960 --> 00:22:48,160 MORE ABOUT NMDA BEFORE I JUMP 583 00:22:48,160 --> 00:22:50,360 INTO THE DELTA RECEPTORS, ALL OF 584 00:22:50,360 --> 00:22:52,280 THIS WHAT I SHOWED YOU WAS IN 585 00:22:52,280 --> 00:22:53,560 THE EXTRA CELLULAR PART BUT OF 586 00:22:53,560 --> 00:22:54,640 COURSE WHAT MOST OF THIS IS 587 00:22:54,640 --> 00:22:56,160 INTERESTED IN IS THE CHANNEL 588 00:22:56,160 --> 00:22:57,560 PART, RIGHT BUT WHEN YOU'RE 589 00:22:57,560 --> 00:23:00,560 DOING FRET, YOU DON'T WANT FLORA 590 00:23:00,560 --> 00:23:03,960 FLORS INSIDE THE CHANNEL SO DREW 591 00:23:03,960 --> 00:23:06,040 STARTED DOING THIS WORK DECIDED 592 00:23:06,040 --> 00:23:08,280 TO PUT THE FLORA FLORS JUST 593 00:23:08,280 --> 00:23:09,320 OUTSIDE OF THE TRANSMEMBRANE 594 00:23:09,320 --> 00:23:12,400 PART TO SEE THE MOTIONS OF THE 595 00:23:12,400 --> 00:23:13,000 TRANSMEMBRANE PART, THE WITH 596 00:23:13,000 --> 00:23:15,800 RESPECT TO WHAT WE ARE SEEING AT 597 00:23:15,800 --> 00:23:17,920 THE LIGAND BINDING DOMAIN 598 00:23:17,920 --> 00:23:19,000 MOTIONS. 599 00:23:19,000 --> 00:23:23,080 SO THIS IS THE APO STATE, AGAIN 600 00:23:23,080 --> 00:23:24,920 IT'S PRIMARILY CLOSED. 601 00:23:24,920 --> 00:23:27,320 I SAY SHORTER, AND WOULD SUGGEST 602 00:23:27,320 --> 00:23:29,600 THAT THE TRANSMEMBRANE PART IS 603 00:23:29,600 --> 00:23:31,520 MORE PACKED AND THE--IN THE 604 00:23:31,520 --> 00:23:33,440 PRESENCE OF GLUTAMATE IS 605 00:23:33,440 --> 00:23:35,360 GLYCINE, IS IT TARTS TO PROBE 606 00:23:35,360 --> 00:23:36,440 SOME MORE DISTANT STATES AND 607 00:23:36,440 --> 00:23:39,720 SUGGESTING THAT MAYBE THAT IS 608 00:23:39,720 --> 00:23:41,240 THE OPEN CHANNEL FORUM AND IN 609 00:23:41,240 --> 00:23:43,680 FACT THE FRACTION THAT IS IN 610 00:23:43,680 --> 00:23:45,880 THESE STATES MATCHES WELL THE 611 00:23:45,880 --> 00:23:46,960 B-FRACTION OF THE CHANNEL THAT'S 612 00:23:46,960 --> 00:23:50,520 OPEN WHEN YOU HAVE BOTH GLYCINE 613 00:23:50,520 --> 00:23:51,120 AND GLUTAMATE. 614 00:23:51,120 --> 00:23:53,480 WE THINK THIS PROBABLY 615 00:23:53,480 --> 00:23:54,400 REPRESENTS THE DESENSITIZED 616 00:23:54,400 --> 00:23:54,760 STATES. 617 00:23:54,760 --> 00:23:55,720 BUT WHAT IS INTERESTING IS THE 618 00:23:55,720 --> 00:23:57,360 FACT THAT YOU SEE LESS 619 00:23:57,360 --> 00:24:00,360 TRANSITIONS BETWEEN THE STATES 620 00:24:00,360 --> 00:24:02,560 IN THE RESTING APO STATE BUT AS 621 00:24:02,560 --> 00:24:08,000 YOU SEE MORE TRANSITIONS IN THE 622 00:24:08,000 --> 00:24:11,360 GLUTAMATE GLYCINE STATES BETWEEN 623 00:24:11,360 --> 00:24:11,960 THE DIFFERENT STATES. 624 00:24:11,960 --> 00:24:14,160 SO IF YOU ARE--IF YOU REMEMBER 625 00:24:14,160 --> 00:24:17,520 IN THE AGONIST BINDING DOMAIN, 626 00:24:17,520 --> 00:24:21,040 THE APO WAS MORE DYNAMIC BUT AS 627 00:24:21,040 --> 00:24:24,400 THE GLUTAMATE AND GLYCINE IS 628 00:24:24,400 --> 00:24:25,720 LESS DYNAMIC AND JUST THE 629 00:24:25,720 --> 00:24:26,880 OPPOSITE HAPPENS THEN IT'S IN 630 00:24:26,880 --> 00:24:28,960 THE TRANSMEMORY RESPONSE BRINE 631 00:24:28,960 --> 00:24:32,160 PART WHERE THE APO STATE OR 632 00:24:32,160 --> 00:24:33,840 CLOSED STATE IS LESS DYNAMIC, IT 633 00:24:33,840 --> 00:24:38,920 LIKES TO BE CLOSED, BUT THE 634 00:24:38,920 --> 00:24:40,360 GLUTAMATE GLYCINE IS MORE 635 00:24:40,360 --> 00:24:41,880 DYNAMIC ALLOWING THE CHANNEL TO 636 00:24:41,880 --> 00:24:42,120 OPEN IT. 637 00:24:42,120 --> 00:24:44,680 SO I THINK ABOUT IT AS A BUNCH 638 00:24:44,680 --> 00:24:45,880 OF ROSES, IF YOU HAVE ROSES AND 639 00:24:45,880 --> 00:24:47,960 YOU HAVE THEM BY THE STEM THEN 640 00:24:47,960 --> 00:24:50,560 THE TOP WILL FLOP OPEN RIGHT? 641 00:24:50,560 --> 00:24:53,000 SO THAT ALLOWS THE STEMS TO BE 642 00:24:53,000 --> 00:24:53,840 CLOSED, THE TRANSMEMBRANE 643 00:24:53,840 --> 00:24:55,960 SEGMENTS TO BE CLOSED WHILE THE 644 00:24:55,960 --> 00:24:59,480 TOP IS FLOPPING,A ROUND ASK JUST 645 00:24:59,480 --> 00:25:02,880 LIKE THE APO STATE. 646 00:25:02,880 --> 00:25:04,760 WHEREAS IN THE OPEN STATE, IF 647 00:25:04,760 --> 00:25:06,760 YOU IMAGINE HOLDING THE BUNCH OF 648 00:25:06,760 --> 00:25:09,880 ROSES WITH JUST THE TOP, THEN 649 00:25:09,880 --> 00:25:11,160 THE BOTTOMS--THE STEMS START TO 650 00:25:11,160 --> 00:25:13,080 MOVE AND THEREFORE THE OTHER 651 00:25:13,080 --> 00:25:14,120 CHANNEL OPENS, OKAY? 652 00:25:14,120 --> 00:25:18,400 SO THIS IS JUST--IT'S NOT JUST 653 00:25:18,400 --> 00:25:19,800 TRUE IN THE RESTING STATE BUT 654 00:25:19,800 --> 00:25:21,720 IT'S TRUE IN THE DESENSITIZED 655 00:25:21,720 --> 00:25:22,920 STATES BUT THE TOP IN THE 656 00:25:22,920 --> 00:25:24,360 CHANNEL IS ABLE TO BE IN A 657 00:25:24,360 --> 00:25:26,800 CLOSED STATE. 658 00:25:26,800 --> 00:25:28,360 SO, WITH THIS BACKGROUND WE THEN 659 00:25:28,360 --> 00:25:34,400 DECIDED TO LOOK AT THIS DATA 660 00:25:34,400 --> 00:25:35,760 RECEPTORS, DELTA RECEPTORS ARE 661 00:25:35,760 --> 00:25:38,960 ORPHAN RECEPTORS AND THAT'S WHAT 662 00:25:38,960 --> 00:25:40,960 THEY'RE CLASSIFIED AS BECAUSE NO 663 00:25:40,960 --> 00:25:43,240 LIGATED ACTIVITY WAS SEEN FOR 664 00:25:43,240 --> 00:25:45,760 THESE DELTA RECEPTORS AND SO 665 00:25:45,760 --> 00:25:51,160 IF--JUST TO SHOW YOU THIS IS HOW 666 00:25:51,160 --> 00:25:53,000 AMPORECEPTORS LOOK BASICALLY YOU 667 00:25:53,000 --> 00:25:54,040 HAVE ACTIVATION AND 668 00:25:54,040 --> 00:25:55,760 DESENSITIZATION AND THEN YOU 669 00:25:55,760 --> 00:25:59,320 APPLY LIGAND, I'M SORRY, AMPO 670 00:25:59,320 --> 00:26:02,000 RECEPTORS FAST AND NMDA 671 00:26:02,000 --> 00:26:03,120 RECEPTORS ARE SLOWER AND CAN YOU 672 00:26:03,120 --> 00:26:04,440 SEE THAT THEY'RE NICELY COUPLED 673 00:26:04,440 --> 00:26:08,000 AT THE TOP AND IF YOU LOOK AT 674 00:26:08,000 --> 00:26:10,760 THE DELTA RECEPTORS WHICH 675 00:26:10,760 --> 00:26:13,840 [INDISCERNIBLE] WHO WAS HERE 676 00:26:13,840 --> 00:26:15,040 THEN WENT ON TO HAVE THE 677 00:26:15,040 --> 00:26:16,160 INDEPENDENT LAB IN INDIA, HE IS 678 00:26:16,160 --> 00:26:20,080 THE 1 WHO DID THE DELTA RECEPTOR 679 00:26:20,080 --> 00:26:20,680 STRUCTURES, AND HE'S--HE SAW 680 00:26:20,680 --> 00:26:25,640 THAT THE TOP IS VERY DECOUPLED, 681 00:26:25,640 --> 00:26:27,040 COMPARED TO THESE 2, RIGHT? 682 00:26:27,040 --> 00:26:29,920 SO LIKE I TOLD YOU THIS SORT OF 683 00:26:29,920 --> 00:26:31,480 REMINDS YOU OF A FLOWERED THING, 684 00:26:31,480 --> 00:26:33,720 IT'S A BUNCH OF ROSES, IT'S 685 00:26:33,720 --> 00:26:34,880 DECOUPLED AND THE WE THINK THE 686 00:26:34,880 --> 00:26:38,400 REASON THERE IS NO ACTIVITY IS 687 00:26:38,400 --> 00:26:40,160 BECAUSE THE CHANNEL IS PROBABLY 688 00:26:40,160 --> 00:26:43,000 DESENSITIZED OR YOU COULD THINK 689 00:26:43,000 --> 00:26:45,760 OF IT AS GLYCINE BINDING DOES 690 00:26:45,760 --> 00:26:46,400 NOT DO ANYTHING. 691 00:26:46,400 --> 00:26:49,960 AND NOW I SAID GLYCINE, AND 692 00:26:49,960 --> 00:26:52,360 YOU'RE LIKE WAIT. THIS IS 693 00:26:52,360 --> 00:26:52,960 GLUTAMATE. 694 00:26:52,960 --> 00:26:55,920 THAT'S THE THING, THE DELTA 695 00:26:55,920 --> 00:26:57,320 RECEPTOR BYPASSING THEY'VE BONE 696 00:26:57,320 --> 00:26:58,960 MD SIMULATIONS AS WELL AS 697 00:26:58,960 --> 00:27:00,920 STRUCTURES AND THEY FOUND IT WAS 698 00:27:00,920 --> 00:27:02,440 NOT APPROPRIATE FOR GLUTAMATE 699 00:27:02,440 --> 00:27:04,880 BINDING, IT WAS PROBABLY GLYCINE 700 00:27:04,880 --> 00:27:06,640 THAT WAS THE LIGAND THAT BOUND 701 00:27:06,640 --> 00:27:09,280 TO THESE RECEPTORS SO EVEN 702 00:27:09,280 --> 00:27:11,160 THOUGH IT'S UNDER GLUTAMATE 703 00:27:11,160 --> 00:27:12,600 RECEPTORS BECAUSE AS YOU CAN SEE 704 00:27:12,600 --> 00:27:14,800 IT HAS VERY SIMILAR STRUCTURE, 705 00:27:14,800 --> 00:27:15,200 RIGHT? 706 00:27:15,200 --> 00:27:18,000 IT HAS--VERY SIMILAR TO THESE 707 00:27:18,000 --> 00:27:18,720 AMPORECEPTORS EXCEPT DECOUPLED 708 00:27:18,720 --> 00:27:32,560 AT THE TOP, SO IT WAS CLASSIFIED 709 00:27:32,560 --> 00:27:36,560 BUT PEOPLE KNOW THAT THESE WERE 710 00:27:36,560 --> 00:27:36,800 BINDING. 711 00:27:36,800 --> 00:27:38,200 BUT NO ACTIVITY WAS SEEN WITH 712 00:27:38,200 --> 00:27:38,680 THE RECEPTORS. 713 00:27:38,680 --> 00:27:39,800 SO WHAT DO THEY DO? 714 00:27:39,800 --> 00:27:41,160 WHY ARE THEY THERE? 715 00:27:41,160 --> 00:27:42,960 BEFORE WE GO ON, I WANTED TO 716 00:27:42,960 --> 00:27:46,200 SHOW YOU SINCE THIS IS A CRYOEM 717 00:27:46,200 --> 00:27:47,080 STRUCTURE 1 STARTS TO WONDER 718 00:27:47,080 --> 00:27:55,960 THAT IT'S JUST IN THE CRYOEM AND 719 00:27:55,960 --> 00:27:57,240 THEN YOU CAN SEE ALL THE STATES 720 00:27:57,240 --> 00:27:59,760 SO WE JUST DID THAT FIRST TO 721 00:27:59,760 --> 00:28:03,080 CONFIRM THAT INDEED IT IS 722 00:28:03,080 --> 00:28:04,760 DECOUPLED AND OUR DATA IS VERY 723 00:28:04,760 --> 00:28:10,000 CLOSE TO WHAT [INDISCERNIBLE] 724 00:28:10,000 --> 00:28:12,280 STRUCTURES SHOWS SO IT IS 725 00:28:12,280 --> 00:28:13,480 SUGGESTING THAT BY ITSELF, THEN 726 00:28:13,480 --> 00:28:15,040 YOU STUDY IT, IT IS REALLY 727 00:28:15,040 --> 00:28:18,040 DECOUPLED LIKE THE STRUCTURE, SO 728 00:28:18,040 --> 00:28:18,480 IT'S NOT THAT. 729 00:28:18,480 --> 00:28:21,360 SO LIKE I SAID, WHAT DO THESE 730 00:28:21,360 --> 00:28:22,760 DO, YOU KNOW WHAT ARE THE 731 00:28:22,760 --> 00:28:25,800 CONTEXT OF THESE DELTA RECEPTORS 732 00:28:25,800 --> 00:28:27,240 IN TERMS OF PHYSIOLOGY MPLET A 733 00:28:27,240 --> 00:28:30,200 LOT OF LABS INCLUDING 734 00:28:30,200 --> 00:28:33,080 [INDISCERNIBLE] LAB HAS TAKEN 735 00:28:33,080 --> 00:28:34,600 THESE DELTA RECEPTORS AND THEY 736 00:28:34,600 --> 00:28:37,240 HAVE SHOWN THAT THEY ACTUALLY 737 00:28:37,240 --> 00:28:41,680 FORM THESE TRANSSYNAPTIC LIKE 738 00:28:41,680 --> 00:28:46,800 CONNECTIONS, SO YOU HAVE NRX, 739 00:28:46,800 --> 00:28:49,200 AND THEN CBLN, AND THEN THE GLUE 740 00:28:49,200 --> 00:28:52,000 AND THEN THEY ALL COME TOGETHER 741 00:28:52,000 --> 00:28:54,400 TO STABILIZE, TO GIVE THE 742 00:28:54,400 --> 00:28:59,480 SYNAPSE A STRUCTURAL STABILITY, 743 00:28:59,480 --> 00:28:59,920 OKAY? 744 00:28:59,920 --> 00:29:03,960 SO IN FACT, IF YOU HAVE SAY IF 745 00:29:03,960 --> 00:29:05,800 ANY OF THESE HAVE MUTATIONS OR 746 00:29:05,800 --> 00:29:09,040 ARE NOT ABLE TO FORM BIG 747 00:29:09,040 --> 00:29:12,480 STRUCTURAL COMPONENTS, THEN YOU 748 00:29:12,480 --> 00:29:15,040 HAVE ISSUES LIKE GATE BECAUSE 749 00:29:15,040 --> 00:29:20,360 GLUD2 IS IN THE CEREBELLUM SO 750 00:29:20,360 --> 00:29:24,360 IT'S INVOLVED IN GAIT ISSUES, OR 751 00:29:24,360 --> 00:29:26,920 IF IT'S IN GLUD1, THEN IT'S 752 00:29:26,920 --> 00:29:29,080 MEMORY SO THIS SORT OF 753 00:29:29,080 --> 00:29:30,560 STRUCTURAL STABILITY IS 754 00:29:30,560 --> 00:29:34,200 EXTREMELY CRITICAL BUT THEN IT 755 00:29:34,200 --> 00:29:36,040 MADE US THINK YOU KNOW IS IT 756 00:29:36,040 --> 00:29:38,920 POSSIBLE THAT HAVING THESE--I 757 00:29:38,920 --> 00:29:43,440 SHOULD ALSO ADD THAT 758 00:29:43,440 --> 00:29:44,960 [INDISCERNIBLE] LAB HAS SHOWN 759 00:29:44,960 --> 00:29:45,960 THAT IT AFFECTS THESE CLUSTERS 760 00:29:45,960 --> 00:29:48,360 AND THERE ARE AT LEAST NRX 761 00:29:48,360 --> 00:29:50,080 MOLECULES THAT ARE IN ANY GIVEN 762 00:29:50,080 --> 00:29:50,360 CLUSTER. 763 00:29:50,360 --> 00:29:54,880 SO IT MADE US THINK THAT THERE'S 764 00:29:54,880 --> 00:29:57,000 A POSSIBLE LED-BASED CLUSTERS 765 00:29:57,000 --> 00:29:57,800 BRINGING THESE DOMAINS TOGETHER 766 00:29:57,800 --> 00:30:00,120 AND IF THAT'S THE CASE COULD 767 00:30:00,120 --> 00:30:01,760 THIS PROTEIN BE A LIGATED 768 00:30:01,760 --> 00:30:02,760 CHANNEL IN THIS PARTICULAR 769 00:30:02,760 --> 00:30:04,240 SITUATION AND AGAIN THIS IS JUST 770 00:30:04,240 --> 00:30:06,360 A CARTOON, NO STRUCTURES ARE 771 00:30:06,360 --> 00:30:08,680 KNOWN OF THE FULL LENGTH OF THIS 772 00:30:08,680 --> 00:30:12,240 COMPLEX, IT'S THE GLUE D, 773 00:30:12,240 --> 00:30:13,120 INDIVIDUAL STRUCTURE IN THE 774 00:30:13,120 --> 00:30:15,320 COMPLEX WITH THE AMINO TERMINAL 775 00:30:15,320 --> 00:30:17,440 DOMAIN, SO THE FULL LENGTH IS 776 00:30:17,440 --> 00:30:19,160 KNOWN, SOME COMPONENTS ARE KNOWN 777 00:30:19,160 --> 00:30:20,480 AND THIS IS A CARTOON JUST SO 778 00:30:20,480 --> 00:30:22,560 THAT YOU KNOW THAT. 779 00:30:22,560 --> 00:30:25,640 SO WHAT WE DECIDED TO DO WAS DO 780 00:30:25,640 --> 00:30:27,560 FULL MEASUREMENTS TO SEE THE 781 00:30:27,560 --> 00:30:28,680 DISTANCE ACROSS THE AMINO 782 00:30:28,680 --> 00:30:30,600 TERMINAL DOMAIN IN THE PRESENCE 783 00:30:30,600 --> 00:30:35,400 AND ABSENCE OF THESE INTERACTING 784 00:30:35,400 --> 00:30:35,680 PROTEINS. 785 00:30:35,680 --> 00:30:37,680 AND OF COURSE INTENSITY BASED 786 00:30:37,680 --> 00:30:39,000 MEASUREMENTS SUGGIEST THAT MAYBE 787 00:30:39,000 --> 00:30:40,800 THE FRET IS MORE IN THE PRESENCE 788 00:30:40,800 --> 00:30:42,360 OF THIS AND NOT IN THE PRESENCE 789 00:30:42,360 --> 00:30:45,120 OF THAT BUT ANYBODY WHO DOES 790 00:30:45,120 --> 00:30:45,600 FLUORESCENCE KNOWS THAT 791 00:30:45,600 --> 00:30:47,040 INTENSITY IS NOT THE WAY TO GO 792 00:30:47,040 --> 00:30:50,200 BECAUSE YOU COULD HAVE OTHER 793 00:30:50,200 --> 00:30:52,880 REASONS FOR THIS SO WE DID 794 00:30:52,880 --> 00:30:54,240 LIFETIME BASE MEASUREMENTS AND 795 00:30:54,240 --> 00:30:57,920 SO THE FILM MEASURES ALSO SHOWED 796 00:30:57,920 --> 00:30:59,360 THAT INDEED THESE AMINO TERMINAL 797 00:30:59,360 --> 00:31:03,360 DOMAIN OFF THE DELTA RECEPTORS 798 00:31:03,360 --> 00:31:04,960 CAME TOGETHER, THEN YOU 799 00:31:04,960 --> 00:31:07,160 HAD--THEN YOU HAD THIS 800 00:31:07,160 --> 00:31:08,560 INTERACTING PROTEINS 801 00:31:08,560 --> 00:31:10,280 NEUREXPECTATIONSIN AND 802 00:31:10,280 --> 00:31:11,040 CEREBELLUM. 803 00:31:11,040 --> 00:31:11,240 OKAY? 804 00:31:11,240 --> 00:31:13,960 SO HAVING THIS INFORMATION THAT 805 00:31:13,960 --> 00:31:15,040 INDEED THESE 2 DO COME TOGETHER, 806 00:31:15,040 --> 00:31:20,160 NOW THE QUESTION IS, CAN IT BE 807 00:31:20,160 --> 00:31:20,760 GATED BY GLYCINE. 808 00:31:20,760 --> 00:31:22,280 SO [INDISCERNIBLE] WHO IS A POST 809 00:31:22,280 --> 00:31:25,000 DOC AND RESEARCH ASSIST APT 810 00:31:25,000 --> 00:31:28,280 PROFESSOR IN MY LAB, DECIDED TO 811 00:31:28,280 --> 00:31:30,360 LACK AT THIS--AT CLUSTERS LIKE 812 00:31:30,360 --> 00:31:32,600 THIS, I KNOW IT'S SORT OF 813 00:31:32,600 --> 00:31:33,520 ANYBODY DOING ELECTROPHYSIOLOGY 814 00:31:33,520 --> 00:31:36,360 KNOWS THIS IS A TERRIBLE WAY OF 815 00:31:36,360 --> 00:31:39,680 DOING EXPERIMENTS, RIGHT? 816 00:31:39,680 --> 00:31:40,960 BECAUSE TIME IS VERY LOW AND IN 817 00:31:40,960 --> 00:31:42,760 FACT THAT IS AT THE BOTTOM OF 818 00:31:42,760 --> 00:31:44,240 THE DISH SO EVEN THOUGH IT LOOKS 819 00:31:44,240 --> 00:31:45,360 LIKE IT'S NOT THIS, IS AT THE 820 00:31:45,360 --> 00:31:47,800 BOTTOM OF THE DISH, SHE JUST 821 00:31:47,800 --> 00:31:49,640 PATCHES A CELL THAT'S SURROUNDED 822 00:31:49,640 --> 00:31:53,560 BY ALL THESE CELLS AND NOW WE 823 00:31:53,560 --> 00:31:56,560 HAVE A GLUE D2 NRX AND 824 00:31:56,560 --> 00:31:57,200 CEREBELLIN EXPRESSED IN THE 825 00:31:57,200 --> 00:31:58,840 CELLS AND WE WANTED TO SEE WHAT 826 00:31:58,840 --> 00:32:01,520 HAPPENS WHEN WE ADD GLYCINE AND 827 00:32:01,520 --> 00:32:05,320 WE GOT THIS REALLY NICE CURRENTS 828 00:32:05,320 --> 00:32:07,400 WHERE GLYCINE ACTIVATES THE 829 00:32:07,400 --> 00:32:10,520 CHANNEL, THIS ACTIVATES THE 830 00:32:10,520 --> 00:32:12,040 CHANNEL, GLUTAMATE DOESN'T DO 831 00:32:12,040 --> 00:32:13,320 MUCH, MAYBE A LITTLE BIT. 832 00:32:13,320 --> 00:32:15,600 WE TRIED A LOT OF OTHER AMINO 833 00:32:15,600 --> 00:32:17,160 ACIDS AND WE DON'T SEE CURRENTS. 834 00:32:17,160 --> 00:32:23,840 BUT I DO HAVE TO TELL YOU, 835 00:32:23,840 --> 00:32:25,240 WARNING, THIS IS VERY DIRTY PREP 836 00:32:25,240 --> 00:32:27,560 AND WE'VE DONE A LOT OF FOLLOW 837 00:32:27,560 --> 00:32:29,360 UP AFTER WE DID THESE 838 00:32:29,360 --> 00:32:31,200 EXPERIMENTS IN THE LAST 9 MONTHS 839 00:32:31,200 --> 00:32:34,360 IT'S BEEN PRETTY MUCH ALL FOLLOW 840 00:32:34,360 --> 00:32:34,560 UPS. 841 00:32:34,560 --> 00:32:38,160 SO WHAT DO I MEAN BY SAYING IT'S 842 00:32:38,160 --> 00:32:39,440 A DIRTY PREP. 843 00:32:39,440 --> 00:32:41,800 FIRST OF ALL AS YOU KNOW, HEK 844 00:32:41,800 --> 00:32:44,080 CELLS ARE TERRIBLE, THEY HAVE A 845 00:32:44,080 --> 00:32:45,520 LOT OF ENDOGENOUS PROTEINS. 846 00:32:45,520 --> 00:32:48,360 WE REALIZE THAT OUR SPECIFIC 847 00:32:48,360 --> 00:32:50,680 BATCH OF HEK CELLS DOESN'T HAVE 848 00:32:50,680 --> 00:32:53,760 ANY GLUE D2, AND IT HAD LITTLE 849 00:32:53,760 --> 00:32:55,800 ENDOGENOUS GLUE D1 AND 2. 850 00:32:55,800 --> 00:32:56,360 WHICH IS FINE. 851 00:32:56,360 --> 00:32:58,160 IF IT WAS THERE IT DOESN'T 852 00:32:58,160 --> 00:32:59,040 AFFECT OUR EXPERIMENTS BECAUSE 853 00:32:59,040 --> 00:33:01,400 YOU'RE NOT DOING MUTATIONAL IS 854 00:33:01,400 --> 00:33:02,360 IT THE STUDIES IN THAT 855 00:33:02,360 --> 00:33:03,520 PARTICULAR SET OF EXPERIMENTS, 856 00:33:03,520 --> 00:33:06,800 BUT THEN, IF YOU WANTED TO STUDY 857 00:33:06,800 --> 00:33:08,320 MUTATIONS IN GLUE D, THEN YOU 858 00:33:08,320 --> 00:33:10,880 NEED A KNOCK OUT OF THE GLUE D 1 859 00:33:10,880 --> 00:33:13,960 AND 2 IN THESE HEK CELLS, AND 860 00:33:13,960 --> 00:33:15,480 DAVID IN HIS LAB HAS MADE THE 861 00:33:15,480 --> 00:33:16,920 KNOCK OUT WHICH IS GREAT SO THAT 862 00:33:16,920 --> 00:33:18,840 EXISTS, SO NOW THIS HAS BECOME 863 00:33:18,840 --> 00:33:20,760 LIKE A--THIS IS THE BEST WAY TO 864 00:33:20,760 --> 00:33:21,400 DO SCIENCE, RIGHT? 865 00:33:21,400 --> 00:33:23,400 BECAUSE A LOT OF LABS ARE ALL 866 00:33:23,400 --> 00:33:24,360 INVOLVED IN DIFFERENT DIRECTIONS 867 00:33:24,360 --> 00:33:27,520 TO MAKE THIS WORK. 868 00:33:27,520 --> 00:33:29,040 BECAUSE WE WANT TO MAKE THIS 869 00:33:29,040 --> 00:33:29,240 WORK. 870 00:33:29,240 --> 00:33:32,920 THEN WHAT WE REALIZED IS THE HEK 871 00:33:32,920 --> 00:33:36,200 CELLS IN THE LAB WAS VERY OLD SO 872 00:33:36,200 --> 00:33:38,600 WE SAID LET'S BUY SOME HEK CELLS 873 00:33:38,600 --> 00:33:41,720 FROM, YOU KNOW ATCC, AND THEN 874 00:33:41,720 --> 00:33:43,360 WHEN CANNED WHAT DID THE CONTROL 875 00:33:43,360 --> 00:33:46,000 EXPERIMENTS SHE STARTED SEEING 876 00:33:46,000 --> 00:33:50,840 CURRENTS AND THEN WE REALIZED 877 00:33:50,840 --> 00:33:53,360 THAT THAT IT TOOK US 9 MONTHS SO 878 00:33:53,360 --> 00:33:54,760 WE WERE TRYING TO FIGURE OUT 879 00:33:54,760 --> 00:33:56,840 WHERE IS THIS COMING FROM AND WE 880 00:33:56,840 --> 00:33:58,120 LOOKED AT TRANSPORTERS BECAUSE 1 881 00:33:58,120 --> 00:34:00,480 OF THE--I WAS TALKING TO A LOT 882 00:34:00,480 --> 00:34:01,840 OF DIFFERENT PEOPLE AND THEY 883 00:34:01,840 --> 00:34:03,240 SUGGESTED MAYBE IT'S A 884 00:34:03,240 --> 00:34:05,800 TRANSPORTER AND WE STARTED DOING 885 00:34:05,800 --> 00:34:06,840 WESTERN BLOTS FOR TRANSPORTERS 886 00:34:06,840 --> 00:34:15,840 AND WE FOUND THAT IN FACT SNAT 887 00:34:15,840 --> 00:34:20,200 2, SODIUM COUPLED NEUTRAL AMINO 888 00:34:20,200 --> 00:34:21,320 ACID TRANSPORTER, NEEDED 889 00:34:21,320 --> 00:34:21,560 CONTROLS. 890 00:34:21,560 --> 00:34:23,160 IF YOU GROW THEM ISOLATED YOU 891 00:34:23,160 --> 00:34:25,440 DON'T GET AS MUCH AS IF YOU GROW 892 00:34:25,440 --> 00:34:27,640 THEM TO FORM THESE CLUSTERS. 893 00:34:27,640 --> 00:34:29,640 SO I'M LEAVING IT OUT THERE FOR 894 00:34:29,640 --> 00:34:31,160 ALL YOUR TRANSPORTER PEOPLE TO 895 00:34:31,160 --> 00:34:37,760 FIGURE OUT WHY, WHAT IS 896 00:34:37,760 --> 00:34:40,640 HAPPENING TO MESS UP OUR GLUE D 897 00:34:40,640 --> 00:34:41,800 DATA? 898 00:34:41,800 --> 00:34:42,560 WHAT IS HAPPENING? 899 00:34:42,560 --> 00:34:44,240 BUT THAT'S ALL I CARE ABOUT. 900 00:34:44,240 --> 00:34:47,760 BUT WE DO HAVE SOME SNAT 1, I 901 00:34:47,760 --> 00:34:49,400 DON'T KNOW IF THIS IS ANYTHING 902 00:34:49,400 --> 00:34:53,560 TO WRITE HOME ABOUT, BUT WE DO 903 00:34:53,560 --> 00:34:59,080 SiRNA, TO GET RID OF SNAT 2. 904 00:34:59,080 --> 00:35:00,800 IN THIS BACKGROUND NOW THAT WE 905 00:35:00,800 --> 00:35:03,360 HAVE LESS CURRENT THIS IS THE 906 00:35:03,360 --> 00:35:05,440 SiRNA TREATED 2 WHERE THE 907 00:35:05,440 --> 00:35:06,200 CURRENTS ARE VERY SMALL SIMILAR 908 00:35:06,200 --> 00:35:08,080 TO WHAT WE'RE SEE NOTHING OUR 909 00:35:08,080 --> 00:35:09,360 PREVIOUS DATA AND THIS IS THE 910 00:35:09,360 --> 00:35:11,200 NEGATIVE TO SHOW THAT YOU KNOW 911 00:35:11,200 --> 00:35:12,920 THIS IS THE SCRAMBLED CONTROL. 912 00:35:12,920 --> 00:35:16,560 IT SHOWS THAT YOU CAN--IT'S NOT 913 00:35:16,560 --> 00:35:19,160 AN SiRNA ISSUE, BUT IT'S 914 00:35:19,160 --> 00:35:21,040 SPECIFIC TO SNAT. 915 00:35:21,040 --> 00:35:23,560 AND THEN ON THAT WE THEN 916 00:35:23,560 --> 00:35:25,040 EXPRESSED GLUE D2 AND CEREBELLUM 917 00:35:25,040 --> 00:35:27,760 AND WERE ABLE TO GET BACK OUR 918 00:35:27,760 --> 00:35:28,680 RESULTS. 919 00:35:28,680 --> 00:35:29,880 SO I THINK IT'S IMPORTANT TO BE 920 00:35:29,880 --> 00:35:32,160 CAREFUL WHEN YOU DO THESE 921 00:35:32,160 --> 00:35:33,680 EXPERIMENTS TO GET A BLANK 922 00:35:33,680 --> 00:35:37,760 BACKGROUND BEFORE YOU START 923 00:35:37,760 --> 00:35:38,560 DOING THESE CONNECTIONS. 924 00:35:38,560 --> 00:35:40,920 FOR SOME REASONS, THE CLUSTERS 925 00:35:40,920 --> 00:35:43,160 DO THINGS TO THESE CELLS AND I 926 00:35:43,160 --> 00:35:44,400 DO NOT KNOW WHAT. 927 00:35:44,400 --> 00:35:45,560 I'M JUST PUTTING IT OUT THERE, 928 00:35:45,560 --> 00:35:48,760 WE ALSO DID THE FIRST 1 AND 2 TO 929 00:35:48,760 --> 00:35:51,080 SHOW THAT YES, IT'S PROBABLY 930 00:35:51,080 --> 00:35:56,080 SNAT 2, THAT'S THE MAIN CULPRIT. 931 00:35:56,080 --> 00:35:58,960 AND YEAH, SO THEN WHILE ALL OF 932 00:35:58,960 --> 00:36:01,600 THIS IS HAPPENING LISA WAS JUST 933 00:36:01,600 --> 00:36:03,680 GETTING VERY FRUSTRATED AND 934 00:36:03,680 --> 00:36:05,440 SAID, YOU KNOW WHAT, I AM DONE 935 00:36:05,440 --> 00:36:06,600 WITH THESE CLUSTER MEASUREMENTS, 936 00:36:06,600 --> 00:36:08,160 WE HAVE TO GET ENOUGH 937 00:36:08,160 --> 00:36:11,800 BACKGROUND, I WILL GO TO A MORE 938 00:36:11,800 --> 00:36:14,560 PURE SYSTEM AND SO SHE ACTUALLY, 939 00:36:14,560 --> 00:36:15,960 YOU NOTICE IT DOESN'T MATTER, AS 940 00:36:15,960 --> 00:36:17,760 LONG AS YOU'RE NOT IN CLUSTERS 941 00:36:17,760 --> 00:36:19,880 YOU DON'T GET CURRENT, IF YOU'RE 942 00:36:19,880 --> 00:36:21,640 NOT IN CLUSTERS IF YOU ARE 943 00:36:21,640 --> 00:36:22,200 MEASURING INDIVIDUAL CELLS, 944 00:36:22,200 --> 00:36:23,720 PEOPLE WHO ARE THE TRANSPORTER 945 00:36:23,720 --> 00:36:25,360 FIELD KNOW THAT IF YOU TAKE 946 00:36:25,360 --> 00:36:29,360 INDIVIDUAL CELLS AND ADD GLYCINE 947 00:36:29,360 --> 00:36:31,160 OR [INDISCERNIBLE], YOU DON'T 948 00:36:31,160 --> 00:36:31,640 GET MUCH CURRENT. 949 00:36:31,640 --> 00:36:33,160 SO SHE SAID I WILL GO TO 950 00:36:33,160 --> 00:36:34,240 INDIVIDUAL CELLS AND SEE HOW I 951 00:36:34,240 --> 00:36:37,120 CAN GET THIS TO WORK? 952 00:36:37,120 --> 00:36:38,280 AND SO THE FIRST THING WE DID 953 00:36:38,280 --> 00:36:41,160 WITH THE INDIVIDUAL CELLS WAS, 954 00:36:41,160 --> 00:36:42,680 OKAY, IF INDEED, FIRST LET'S 955 00:36:42,680 --> 00:36:44,120 VERIFY IF YOU CAN CROSS LINK THE 956 00:36:44,120 --> 00:36:46,320 TOP AND GET CURRENTS SO WE DID 957 00:36:46,320 --> 00:36:48,960 THE CHEMICAL METHOD OF DOING 958 00:36:48,960 --> 00:36:50,440 STUFF. 959 00:36:50,440 --> 00:36:53,160 SO SHE USED, SOPHISTICATED WE 960 00:36:53,160 --> 00:36:53,960 HAVE BASICALLY CYSTINES 961 00:36:53,960 --> 00:36:57,440 INTRODUCED ON BOTH SIDES AND WE 962 00:36:57,440 --> 00:36:59,520 USED BI FUNCTIONAL CYSTINE CROSS 963 00:36:59,520 --> 00:37:00,800 LINKERS OF VARIOUS LENGTHS AND 964 00:37:00,800 --> 00:37:03,920 YOU SEE THAT WHEN YOU USE A SHOT 965 00:37:03,920 --> 00:37:05,680 CROSS LINKER, YOU GET--YOU GET 966 00:37:05,680 --> 00:37:09,680 CURRENTS AND THEN WHEN YOU USE 967 00:37:09,680 --> 00:37:10,840 LONG CROSS LINKER, THIS IS 968 00:37:10,840 --> 00:37:13,160 ROUGHLY THE LINK THAT IS THERE 969 00:37:13,160 --> 00:37:15,960 IN THE CRYO EM STRUCTURES, IT'S 970 00:37:15,960 --> 00:37:17,200 ABOUT 21-ANGSTROMS SO WHEN YOU 971 00:37:17,200 --> 00:37:20,080 GET TO 25, YOU DON'T SEE ANY 972 00:37:20,080 --> 00:37:22,720 CURRENTS SO THIS CRYO EM 973 00:37:22,720 --> 00:37:24,520 STRUCTURE IS PROBABLY THE 974 00:37:24,520 --> 00:37:25,200 DESENSITIZED STATE. 975 00:37:25,200 --> 00:37:28,520 AND OF COURSE, YOU WANT TO, ALL 976 00:37:28,520 --> 00:37:30,040 OF THIS MEANS NOTHING BECAUSE 977 00:37:30,040 --> 00:37:32,120 YOU COULD HAVE DIFFERENT AMOUNTS 978 00:37:32,120 --> 00:37:34,280 OF PROTEIN BEING EXPRESSED SO 979 00:37:34,280 --> 00:37:36,560 SHE DID ELEMENTS TO LOOK AT 980 00:37:36,560 --> 00:37:37,560 PROBABILITY OF OPENING AND YES, 981 00:37:37,560 --> 00:37:39,920 THIS IS MORE OPEN. 982 00:37:39,920 --> 00:37:41,360 BUT THEN THE FINAL SET OF 983 00:37:41,360 --> 00:37:43,360 EXPERIMENTS SHE DID IS SHE SAID, 984 00:37:43,360 --> 00:37:48,160 OKAY, I'M GOING TO EXPRESS GLUD2 985 00:37:48,160 --> 00:37:56,440 IN THESE CELLS AND WE WILL 986 00:37:56,440 --> 00:38:00,520 PURIFY AND SO THAT IT'S SORT OF 987 00:38:00,520 --> 00:38:03,560 AND ON THEN ADD THIS ON TOLET 988 00:38:03,560 --> 00:38:08,280 PIPETTE, THE CEREBELLUM ALONG 989 00:38:08,280 --> 00:38:09,800 WITH GLIESINE AND THEN ACTIVATE 990 00:38:09,800 --> 00:38:11,320 THE CHANNELS AT THE SINGLE 991 00:38:11,320 --> 00:38:12,760 CHANNEL MODE. 992 00:38:12,760 --> 00:38:15,920 SO GLUE D2 ALONE WHEN YOU HAD 993 00:38:15,920 --> 00:38:18,800 GLYCINE IN THE PIPETTE, YOU GET 994 00:38:18,800 --> 00:38:21,160 NO SINGLE CHABLES WHEN YOU ADD 995 00:38:21,160 --> 00:38:22,000 CEREBELLUM WITH GLYCINE, YOU 996 00:38:22,000 --> 00:38:24,200 DON'T SEE MUCH, BUT IF WE RECORD 997 00:38:24,200 --> 00:38:27,040 FOR LONG ENOUGH TIME WE ACTUALLY 998 00:38:27,040 --> 00:38:28,520 SEE 1 OR 2 SMALL LENGTHS, I WANT 999 00:38:28,520 --> 00:38:30,840 TO ADD THAT THERE, BUT THEN YOU 1000 00:38:30,840 --> 00:38:34,720 ADD ALL 3, YOU START SEEING 1001 00:38:34,720 --> 00:38:35,160 OPENINGS. 1002 00:38:35,160 --> 00:38:35,360 OKAY? 1003 00:38:35,360 --> 00:38:38,640 AND SINCE THIS TIME, SHE HAS 1004 00:38:38,640 --> 00:38:41,760 ACTUALLY PURIFIED, A GLUE D 2 1005 00:38:41,760 --> 00:38:43,680 ALSO AND DIP MEASUREMENTS THAT 1006 00:38:43,680 --> 00:38:47,920 YOU HAVE GLUE D2 ASK CBLN 1007 00:38:47,920 --> 00:38:49,400 ALTOGETHER AND LOOK AT THE 1008 00:38:49,400 --> 00:38:51,960 CHANNELS IN BILAYERS AND SHE 1009 00:38:51,960 --> 00:38:53,280 SEES SINGLE CHANNELS WHEN SHE 1010 00:38:53,280 --> 00:38:55,360 HAS ALL 3 COMPONENTS. 1011 00:38:55,360 --> 00:38:57,360 THIS IS AS OF LAST WEEK SO I 1012 00:38:57,360 --> 00:38:59,960 DIDN'T INCLUDE THAT DATA YET. 1013 00:38:59,960 --> 00:39:02,520 SO SO THE NEXT FINAL PART WAS WE 1014 00:39:02,520 --> 00:39:04,680 WANTED TO MAKE SURE THAT WHAT WE 1015 00:39:04,680 --> 00:39:08,160 SAW IN THE SINGLE CHANNEL 1016 00:39:08,160 --> 00:39:16,560 RECORDING WAS REFLECTED IN OUR 1017 00:39:16,560 --> 00:39:17,960 SINGLE MOLECULE THREAT. 1018 00:39:17,960 --> 00:39:22,080 SO AND THE TERMINAL DOMAIN 1019 00:39:22,080 --> 00:39:23,040 ADDING NEW CEREBELLIN AND 1020 00:39:23,040 --> 00:39:26,320 COMPLEX TO SEE WHAT HAPPENS AND 1021 00:39:26,320 --> 00:39:29,280 THIS IS WITHOUT NRX AND 1022 00:39:29,280 --> 00:39:30,560 CEREBELLUM, THIS IS THE RAW 1023 00:39:30,560 --> 00:39:34,480 DATA, THIS IS THE HISTOGRAM 1024 00:39:34,480 --> 00:39:37,080 WITHOUT NRX AND CEREBELLIN AND 1025 00:39:37,080 --> 00:39:45,520 YOU CAN CLEARLY SEE THAT THE TOP 1026 00:39:45,520 --> 00:39:46,560 COMES TOGETHER. 1027 00:39:46,560 --> 00:39:49,120 SO THOSE RECEPTORS ARE NOT 1028 00:39:49,120 --> 00:39:50,960 ORPHAN RECEPTORS, THEY ARE 1029 00:39:50,960 --> 00:39:53,560 ACTUALLY GLUE MARIOUS SEEN 1030 00:39:53,560 --> 00:39:54,000 NEGATED CHANNELS. 1031 00:39:54,000 --> 00:39:57,000 THE ROLE IN TERMS OF PHYSIOLOGY 1032 00:39:57,000 --> 00:40:00,360 IS, WE STILL DON'T KNOW WHAT 1033 00:40:00,360 --> 00:40:11,360 THAT GLYCINE AND DC CURRENTS DO, 1034 00:40:11,360 --> 00:40:19,160 AND AND BEYOND THAT, BUT 1035 00:40:19,160 --> 00:40:20,600 CLEARLY, IT IS BOUND TO THESE, 1036 00:40:20,600 --> 00:40:24,560 IT SEEMS THAT YOU CAN GET THESE 1037 00:40:24,560 --> 00:40:24,840 CHANNELS. 1038 00:40:24,840 --> 00:40:26,720 AND I'M GOING TO END WITH THAT 1039 00:40:26,720 --> 00:40:33,800 AND JUST THANK ALL THE PEOPLE 1040 00:40:33,800 --> 00:40:38,000 WHO WORKED ON THIS, GISTY HELPED 1041 00:40:38,000 --> 00:40:40,240 US GET STARTED TO THE SINGLE 1042 00:40:40,240 --> 00:40:42,200 MOLECULE 38 AND ALEX IS OUR 1043 00:40:42,200 --> 00:40:43,280 COLLABORATOR ON MD SIMULATION, 1044 00:40:43,280 --> 00:40:48,240 SO WITH THAT I WILL STOP. 1045 00:40:48,240 --> 00:40:55,960 [ APPLAUSE ] 1046 00:40:55,960 --> 00:41:01,440 >> SO I THINK FOR THE NRX, 1047 00:41:01,440 --> 00:41:03,040 SHOWED US [INDISCERNIBLE] DO YOU 1048 00:41:03,040 --> 00:41:03,320 NEED BOTH? 1049 00:41:03,320 --> 00:41:05,960 OR IF YOU ADD 1 OR THE OTHER BY 1050 00:41:05,960 --> 00:41:08,600 ITSELF [INDISCERNIBLE]? 1051 00:41:08,600 --> 00:41:12,640 >> YEAH, THIS IS JUST WITH CBLN, 1052 00:41:12,640 --> 00:41:14,880 AND WE'VE ALSO DONE NRX ALONE. 1053 00:41:14,880 --> 00:41:17,360 THE TOP 1 IS THE CBLN, NO IT 1054 00:41:17,360 --> 00:41:18,200 DOESN'T, YOU NEED BOTH. 1055 00:41:18,200 --> 00:41:20,760 I TAKE IT BACK. 1056 00:41:20,760 --> 00:41:22,600 SO CBLN ALONE LIKE I SAID, ONCE 1057 00:41:22,600 --> 00:41:24,800 IN A WHILE YOU SEE THESE BLIPS 1058 00:41:24,800 --> 00:41:28,400 BUT I DON'T KNOW IT COULD JUST 1059 00:41:28,400 --> 00:41:29,480 SOME RANDOM BACKGROUND ACTIVITY 1060 00:41:29,480 --> 00:41:33,960 BUT WE DON'T SEE THAT AS OFTEN 1061 00:41:33,960 --> 00:41:37,120 WITH GLUD ALONE BECAUSE IT HAS 1062 00:41:37,120 --> 00:41:38,080 THE ABILITY TO CROSS. 1063 00:41:38,080 --> 00:41:44,800 >> DO YOU THINK THAT THEY'RE 1064 00:41:44,800 --> 00:41:45,640 MAKING [INDISCERNIBLE]? 1065 00:41:45,640 --> 00:41:47,840 >> NRX IS THE 1 THAT'S 1066 00:41:47,840 --> 00:41:48,680 MAKING--THE CLUSTERS IS ABLE TO 1067 00:41:48,680 --> 00:41:51,240 DO THAT AND WE HAVE NRX DIMER 1068 00:41:51,240 --> 00:41:53,000 WHICH WE HAVE MADE FROM 1069 00:41:53,000 --> 00:41:54,760 COILED--IT'S NOT A DIMER--I 1070 00:41:54,760 --> 00:41:57,080 DON'T THINK IT'S A DIMER. 1071 00:41:57,080 --> 00:41:57,600 I DON'T KNOW. 1072 00:41:57,600 --> 00:42:00,000 AT LEAST WE DON'T KNOW IT'S A 1073 00:42:00,000 --> 00:42:01,480 DIMER IN PHYSIOLOGY, IT'S NRX BY 1074 00:42:01,480 --> 00:42:04,480 ITSELF, SO WE MADE A COIL COIL 1075 00:42:04,480 --> 00:42:06,360 NRX DIMER WHICH IS WHAT REALLY 1076 00:42:06,360 --> 00:42:28,440 MAKES THE ACTIVITY, YEAH. 1077 00:42:28,440 --> 00:42:29,480 >> REALLY INTERESTING WORK DO 1078 00:42:29,480 --> 00:42:31,360 YOU KNOW ANYTHING ABOUT 1079 00:42:31,360 --> 00:42:31,720 [INDISCERNIBLE]? 1080 00:42:31,720 --> 00:42:34,320 >> YES, SO IT CAN--AGAIN, 1081 00:42:34,320 --> 00:42:34,960 EVERYBODY'S VERY PRELIMINARY, 1082 00:42:34,960 --> 00:42:36,600 ALL THIS HAPPENED OVER THE LAST 1083 00:42:36,600 --> 00:42:38,240 FEW YEARS. 1084 00:42:38,240 --> 00:42:41,800 SO SHE'S DONE--IT'S OF COURSE 1085 00:42:41,800 --> 00:42:44,160 SODIUM, POTASSIUM, POTASSIUM CAN 1086 00:42:44,160 --> 00:42:46,160 GO THROUGH AND THAT SHE DID THE 1087 00:42:46,160 --> 00:42:48,040 POTASSIUM BECAUSE WE WANTED TO 1088 00:42:48,040 --> 00:42:50,520 GET RID OF THE SNAT, WHICH WAS 1089 00:42:50,520 --> 00:42:51,960 KILLING US AND THEN CALCIUM, 1090 00:42:51,960 --> 00:42:54,680 THERE IS SOME, BUT IF YOU ADD 1091 00:42:54,680 --> 00:42:57,960 LIKE ZINC FOR INSTANCE IT 1092 00:42:57,960 --> 00:43:02,800 INHIBITS THE CHANNEL. 1093 00:43:02,800 --> 00:43:03,600 >> [INDISCERNIBLE] 1094 00:43:03,600 --> 00:43:03,840 >> YES. 1095 00:43:03,840 --> 00:43:05,000 WE WE'VE TRIED THEM. 1096 00:43:05,000 --> 00:43:06,760 SO IT'S VERY LITTLE. 1097 00:43:06,760 --> 00:43:09,640 NO IT'S NOT--IT'S NOT LARGE, SO 1098 00:43:09,640 --> 00:43:10,600 THAT IS DEFINITELY ON THE LIST 1099 00:43:10,600 --> 00:43:12,000 OF EXPERIMENTS TO DO. 1100 00:43:12,000 --> 00:43:13,400 WE REALLY FEEL LIKE THAT WOULD 1101 00:43:13,400 --> 00:43:14,600 REALLY HELP US AND NOW THAT 1102 00:43:14,600 --> 00:43:16,440 WE'VE GOTTEN RID OF ALL OTHER 1103 00:43:16,440 --> 00:43:18,000 BACKGROUNDS, SO I THINK IT WILL 1104 00:43:18,000 --> 00:43:26,920 BE EASIER TO SEE THE CALCIUM. 1105 00:43:26,920 --> 00:43:27,120 YEAH. 1106 00:43:27,120 --> 00:43:27,600 >> RELATED QUESTION. 1107 00:43:27,600 --> 00:43:30,520 ARE THERE ANY OTHER 1108 00:43:30,520 --> 00:43:33,040 [INDISCERNIBLE] RECEPTOR? 1109 00:43:33,040 --> 00:43:33,880 >> OH YEAH. 1110 00:43:33,880 --> 00:43:34,200 ABSOLUTELY. 1111 00:43:34,200 --> 00:43:35,840 SORRY, I DONT WANT TO GO INTO IT 1112 00:43:35,840 --> 00:43:37,800 BUT WHEN I SAID THE PILLARS, 1113 00:43:37,800 --> 00:43:40,160 THAT'S A GOOD POINT BECAUSE MOST 1114 00:43:40,160 --> 00:43:43,520 OF THE WORK HAS FOCUSED ON THE 1115 00:43:43,520 --> 00:43:44,680 SECONDARY MESSENGERS PATHWAY 1116 00:43:44,680 --> 00:43:47,800 BECAUSE THE C-TERMINAL DOMAIN IS 1117 00:43:47,800 --> 00:43:48,800 EXTREMELY CRITICAL IN MEDIATING 1118 00:43:48,800 --> 00:43:49,600 THAT PATHWAY AND EVEN THERE, 1119 00:43:49,600 --> 00:43:52,360 THERE IS A BIT OF A CONTROVERSY 1120 00:43:52,360 --> 00:43:54,200 WHERE SOME PEOPLE THINK GLYCINE 1121 00:43:54,200 --> 00:43:57,000 IS NECESSARY TO MEDIATE THE 1122 00:43:57,000 --> 00:43:59,120 C-TERMINAL IMMEDIATE AND 1123 00:43:59,120 --> 00:43:59,600 INTRACELLULAR SIGNALING 1124 00:43:59,600 --> 00:44:00,160 PATHWAYS. 1125 00:44:00,160 --> 00:44:01,800 OTHERS SAY I DON'T NEED ANY OF 1126 00:44:01,800 --> 00:44:02,160 THIS. 1127 00:44:02,160 --> 00:44:04,000 ALL YOU NEED IS--THE LAB SAYS 1128 00:44:04,000 --> 00:44:06,240 ALL YOU NEED IS THE AMINO 1129 00:44:06,240 --> 00:44:07,320 TERMINAL DOMAIN MAKING THIS 1130 00:44:07,320 --> 00:44:10,280 ACROSS, AS LONG AS YOU HAVE SOME 1131 00:44:10,280 --> 00:44:11,800 PROTEIN CONNECTING THE 1132 00:44:11,800 --> 00:44:13,400 C-TERMINUS TO THE AMINO TERMINAL 1133 00:44:13,400 --> 00:44:15,040 DOMAIN, IT'S ALL FINE. 1134 00:44:15,040 --> 00:44:16,720 LTP AND STILL FINE. 1135 00:44:16,720 --> 00:44:18,520 ABOUT YOU OUR OTHER LABS SHOW 1136 00:44:18,520 --> 00:44:20,120 THAT IF YOU MUTED THE GLUE 1137 00:44:20,120 --> 00:44:22,200 MARIOUS SEEN BINDING SITE FOR 1138 00:44:22,200 --> 00:44:23,640 INSTANCE THEN LTP IS DISRUPTIVE 1139 00:44:23,640 --> 00:44:28,520 SO THEY'RE LIKE, NO, GLYCINE 1140 00:44:28,520 --> 00:44:29,800 BIND SUGGEST IMPORTANT BUT THEY 1141 00:44:29,800 --> 00:44:32,280 SAID IT AFFECTS THE C-TERMINAL 1142 00:44:32,280 --> 00:44:33,600 DOMAIN, NOTHING TO DO WITH 1143 00:44:33,600 --> 00:44:35,640 CHANNEL ACTIVITY SO NOW WE'RE 1144 00:44:35,640 --> 00:44:36,840 TRYING TO--BUT THE MUTANT WAS 1145 00:44:36,840 --> 00:44:38,960 MADE BUT THE KNOCK OUT WAS KNOCK 1146 00:44:38,960 --> 00:44:41,480 OUT AND KNOCK-IN OF THAT MUTANT 1147 00:44:41,480 --> 00:44:43,440 CHANNEL MUTANT WAS MADE BUT WE 1148 00:44:43,440 --> 00:44:45,000 DID SEE A SMALL CHANGE IN 1149 00:44:45,000 --> 00:44:46,600 BEHAVIOR BUT THEY SAID IT'S NOT 1150 00:44:46,600 --> 00:44:47,960 THAT SIGNIFICANT AND I AGREE, 1151 00:44:47,960 --> 00:44:49,520 THAT'S NOT THE MAIN THING BUT I 1152 00:44:49,520 --> 00:44:51,640 THINK THE CHANNEL ACTIVITY IS 1153 00:44:51,640 --> 00:44:51,840 THERE. 1154 00:44:51,840 --> 00:44:53,440 IT MAY NOTHING LIKE THAT BE THE 1155 00:44:53,440 --> 00:44:56,160 MAIN POINT. 1156 00:44:56,160 --> 00:44:59,920 IT'S THERE. 1157 00:44:59,920 --> 00:45:00,400 YEAH. 1158 00:45:00,400 --> 00:45:03,200 >> CAN YOU REPEAT THE QUESTION 1159 00:45:03,200 --> 00:45:06,000 FOR THE VIDEOCAST AUDIENCE. 1160 00:45:06,000 --> 00:45:09,400 >> WHEN COMPARING THESE IN THE 1161 00:45:09,400 --> 00:45:11,120 ALSTERRERY DOMAIN THAT SHE'S THE 1162 00:45:11,120 --> 00:45:16,280 SEQUENCE FOR EVOLUTIONARY 1163 00:45:16,280 --> 00:45:16,600 SIGNAL 1164 00:45:16,600 --> 00:45:16,840 DIFFER? 1165 00:45:16,840 --> 00:45:19,200 >> I THINK THAT WOULD BE A GREAT 1166 00:45:19,200 --> 00:45:21,760 COLLABORATION WITH LUCY. 1167 00:45:21,760 --> 00:45:25,080 >> AND THEN I HAD A QUESTION IF 1168 00:45:25,080 --> 00:45:26,640 YOU CAN REPEAT THE QUESTION IN 1169 00:45:26,640 --> 00:45:27,720 YOUR MIC, DO I UNDERSTAND THIS 1170 00:45:27,720 --> 00:45:37,280 THAT IN THIS CASE, YOU HAVE THE 1171 00:45:37,280 --> 00:45:42,560 DELTA RECEPTOR SMA, EXTRACTED. 1172 00:45:42,560 --> 00:45:45,920 >> OKAY, THIS WAS DONE ON CELLS, 1173 00:45:45,920 --> 00:45:51,600 AND THE SMA PURIFIED NRX 1174 00:45:51,600 --> 00:45:56,440 WAS--BECAUSE WHEN WE USE 1175 00:45:56,440 --> 00:45:58,080 DETERGENT PURIFIED NRX AND THE 1176 00:45:58,080 --> 00:46:00,400 PURIFIED WASN'T STABLE THAT'S 1177 00:46:00,400 --> 00:46:04,040 WHY WE WENT WITH PURIFIED NRX 1178 00:46:04,040 --> 00:46:06,400 INSTEAD OF CEREBELLIN. 1179 00:46:06,400 --> 00:46:08,000 >> [INDISCERNIBLE] WITH BOTH 1180 00:46:08,000 --> 00:46:09,480 PROTEINS IN THE EXTRACTED 1181 00:46:09,480 --> 00:46:09,800 DEPARTMENT. 1182 00:46:09,800 --> 00:46:10,360 >> WE'RE TRYING. 1183 00:46:10,360 --> 00:46:11,400 >> THANK YOU VERY MUCH. 1184 00:46:11,400 --> 00:46:11,920 >> IT'S THERE. 1185 00:46:11,920 --> 00:46:19,600 IT'S BEING DONE AS WE ARE 1186 00:46:19,600 --> 00:46:20,000 SPEAKING. 1187 00:46:20,000 --> 00:46:23,400 NOT JUST BY OUR LABS, SEVERAL 1188 00:46:23,400 --> 00:46:23,880 LABS. 1189 00:46:23,880 --> 00:46:24,960 SO A LOT IN THIS FIELD AT THIS 1190 00:46:24,960 --> 00:46:25,920 POINT BECAUSE A LOT OF PEOPLE 1191 00:46:25,920 --> 00:46:38,800 ARE WORKING ON THIS. 1192 00:46:38,800 --> 00:46:41,200 >> WELL, IF THERE ARE NO OTHER 1193 00:46:41,200 --> 00:46:42,160 QUESTIONS, THEN LET'S JOIN IN 1194 00:46:42,160 --> 00:46:44,600 THANKING OUR SPEAKER AGAIN FOR A 1195 00:46:44,600 --> 00:46:45,440 WONDERFUL PRESENTATION. 1196 00:46:45,440 --> 00:48:09,200 [ APPLAUSE ]