Methods in enzymology, volume 556

METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N ABELSON and MELVIN I SIMON Division of Biology California Institute of Technology Pasadena, California ANNA MARIE PYLE Departments of Molecular, Cellular and Developmental Biology and Department of Chemistry Investigator Howard Hughes Medical Institute Yale University Founding Editors SIDNEY P COLOWICK and NATHAN O KAPLAN Academic Press is an imprint of Elsevier 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101–4495, USA 125 London Wall, London, EC2Y 5AS, UK The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2015 Copyright © 2015 Elsevier Inc All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher Details on how to seek permission, further information about the 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contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein ISBN: 978-0-12-801521-6 ISSN: 0076-6879 For information on all Academic Press publications visit our website at store.elsevier.com CONTRIBUTORS Roslin J Adamson Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom Susana Andrade Institute for Biochemistry, and BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg im Breisgau, Germany Tonia Aristotelous Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom Aidin R Balo Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Jeffrey M Becker Microbiology Department, University of Tennessee Imre Berger School of Biochemistry, University of Bristol, Bristol, United Kingdom; European Molecular Biology Laboratory, and Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Unite´ mixte de Recherche, Grenoble, France Frank Bernhard Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W Goethe-University, Frankfurt-am-Main, Germany Nicolas Bertheleme Department of Life Sciences, Imperial College London, London, United Kingdom Rajinder P Bhullar Department of Oral Biology, College of Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada Kory M Blocker Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, Louisiana, USA Christoph Boes The Medical Research Council, Mitochondrial Biology Unit, Cambridge, United Kingdom Mathieu Botte European Molecular Biology Laboratory, and Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Unite´ mixte de Recherche, Grenoble, France Zachary T Britton Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA xv xvi Contributors Bernadette Byrne Department of Life Sciences, Imperial College London, London, United Kingdom Nico Callewaert Unit of Medical Biotechnology, Department of Medical Protein Research; Inflammation Research Center, VIB-UGhent, and Department of Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium Lydia N Caro Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Raja Chakraborty Department of Oral Biology, College of Dentistry, and Biology of Breathing Group, Children’s Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada Prashen Chelikani Department of Oral Biology, College of Dentistry, and Biology of Breathing Group, Children’s Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada Katrien Claes Unit of Medical Biotechnology, Department of Medical Protein Research; Inflammation Research Center, VIB-UGhent, and Department of Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium Benjamin Cle´menc¸on Institute of Biochemistry and Molecular Medicine (IBMM), and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland Ian Collinson School of Biochemistry, University of Bristol, Bristol, United Kingdom Patricia M Dijkman Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom Simon Dowell Department of Molecular Discovery Research, GlaxoSmithKline, Hertfordshire, United Kingdom Volker D€ otsch Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W Goethe-University, Frankfurt-am-Main, Germany Ashvini K Dubey National Centre for Biological Sciences, TIFR, Bangalore, and Department of Biotechnology, University of Mysore, Mysore, India Contributors xvii Oliver Einsle Institute for Biochemistry, and BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg im Breisgau, Germany Matthias Elgeti Institut f€ ur Medizinische Physik und Biophysik (CC2), Charite-Universitaătsmedizin Berlin, Berlin, Germany Oliver P Ernst Department of Biochemistry, and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Klaus Fendler Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Frankfurt am Main, Germany James D Fessenden Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA Michael Fine Institute of Biochemistry and Molecular Medicine (IBMM), and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland Eshan Ghosh Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India Ashwini Godbole National Centre for Biological Sciences, TIFR, Bangalore, India Alan D Goddard School of Life Sciences, University of Lincoln, Lincoln, United Kingdom Adrian Goldman Department of Biochemistry, Helsinki University, Helsinki, Finland, and School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom Mouna Guerfal Unit of Medical Biotechnology, Department of Medical Protein Research; Inflammation Research Center, VIB-UGhent, and Department of Biochemistry and Microbiology, Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium Yvonne Hackmann Biochemistry Center, Heidelberg University, Heidelberg, Germany Matthias A Hediger Institute of Biochemistry and Molecular Medicine (IBMM), and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland xviii Contributors Erik Henrich Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W Goethe-University, Frankfurt-am-Main, Germany Peter W Hildebrand Institut f€ ur Medizinische Physik und Biophysik (CC2), Charite-Universitaătsmedizin Berlin, Berlin, Germany, and AG ProteInformatics Franz Y Ho Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands Klaus Peter Hofmann Institut f€ ur Medizinische Physik und Biophysik (CC2), Charite-Universitaătsmedizin Berlin, and Zentrum f ur Biophysik und Bioinformatik, Humboldt-Universitaăt zu Berlin, Berlin, Germany Andrew L Hopkins Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom Veli-Pekka Jaakola Novartis Institutes of Biomedical Research, Basel, Switzerland Lisa Joedicke Biochemistry Center, Heidelberg University, Heidelberg, Germany Zachary Lee Johnson Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA Martin S King The Medical Research Council, Mitochondrial Biology Unit, Cambridge, United Kingdom Joanna Komar School of Biochemistry, University of Bristol, Bristol, United Kingdom Punita Kumari Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India Edmund R.S Kunji The Medical Research Council, Mitochondrial Biology Unit, Cambridge, United Kingdom Wei L€ u Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Freiburg im Breisgau, Germany Michael Lafontaine Department of Structural Biology, Institute of Biophysics and Center of Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany Contributors xix C Roy D Lancaster Department of Structural Biology, Institute of Biophysics and Center of Human and Molecular Biology (ZHMB), Saarland University, Homburg, Germany Seok-Yong Lee Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA Zhijie Li Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Kenneth Lundstrom PanTherapeutics, Lutry, Switzerland Mohana Mahalingam Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA M.K Mathew National Centre for Biological Sciences, TIFR, Bangalore, India Patrick M McNeely Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA Rohan Mitra National Centre for Biological Sciences, TIFR, Bangalore, India Christophe J Moreau Institut de Biologie Structurale (IBS), University of Grenoble Alpes; CNRS, IBS, LabEx ICST, and CEA, IBS, Grenoble, France Fred Naider Chemistry Department, College of Staten Island, City University of New York Andrea N Naranjo Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA Iva Navratilova Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom Kumari Nidhi Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India Katarzyna Niescierowicz Institut de Biologie Structurale (IBS), University of Grenoble Alpes; CNRS, IBS, LabEx ICST, and CEA, IBS, Grenoble, France Chikwado A Opefi Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom xx Contributors Vale´rie Panneels Biochemistry Center, Heidelberg University, Heidelberg, Germany Bert Poolman Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands Palakolanu S Reddy National Centre for Biological Sciences, TIFR, Bangalore, India Philip J Reeves Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom Rosana Ina´cio dos Reis Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom Jean Revilloud Institut de Biologie Structurale (IBS), University of Grenoble Alpes; CNRS, IBS, LabEx ICST, and CEA, IBS, Grenoble, France James M Rini Department of Biochemistry, and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Anne S Robinson Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, Louisiana, and Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA Adriana Rycovska-Blume Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Frankfurt am Main, Germany Tuulia Saarenpaăaă Department of Biochemistry, Helsinki University, Helsinki, Finland Christiane Schaffitzel School of Biochemistry, University of Bristol, Bristol, United Kingdom; European Molecular Biology Laboratory, and Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Unite´ mixte de Recherche, Grenoble, France Patrick Scheerer Institut f ur Medizinische Physik und Biophysik (CC2), Charite-Universitaătsmedizin Berlin, Berlin, Germany, and AG Protein X-ray Crystallography & Signal Transduction Philipp Schneider Institute of Biochemistry and Molecular Medicine (IBMM), and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland Arun K Shukla Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India Contributors xxi Shweta Singh Department of Life Sciences, Imperial College London, London, United Kingdom Irmgard Sinning Biochemistry Center, Heidelberg University, Heidelberg, Germany Steven O Smith Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA Martha E Sommer Institut f ur Medizinische Physik und Biophysik (CC2), Charite-Universitaătsmedizin Berlin, Berlin, Germany Randy B Stockbridge Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts, USA Michal Szczepek Institut f ur Medizinische Physik und Biophysik (CC2), Charite-Universitaătsmedizin Berlin, Berlin, Germany Dale Tranter Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom Ming-Feng Tsai Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts, USA Ned Van Eps Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Michel Vivaudou Institut de Biologie Structurale (IBS), University of Grenoble Alpes; CNRS, IBS, LabEx ICST, and CEA, IBS, Grenoble, France Anthony Watts Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom Bing Xu Department of Oral Biology, College of Dentistry, and Biology of Breathing Group, Children’s Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada Carissa L Young Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA PREFACE Integral membrane proteins constitute a significant portion of the entire proteome in different organisms They mediate a wide range of signal recognition and communication processes across the cell membranes These proteins are one of the most important classes of drug targets, and more than half of the currently marketed drugs are aimed at membrane proteins In spite of their crucial physiological roles, structural characterization, especially high-resolution structure determination, of membrane proteins lags significantly behind that of soluble proteins There are numerous challenges encountered at every step in the process of membrane protein crystallography such as recombinant protein expression, homogenous purification, and crystallization These two volumes of Methods in Enzymology aim to provide a comprehensive coverage of various steps involved in the process of membrane protein structural characterization through general protocols and case examples The very first step in the process of structural studies of membrane proteins is their recombinant expression in heterologous hosts for large-scale protein production In Section I of Volume I, we provide a collection of chapters that describe either a step-by-step protocol or a general overview for recombinant expression of various types of membrane proteins in different expression hosts These chapters cover recent advances in conventional E coli-based expression of membrane proteins, yeast-based expression systems for large-scale production of eukaryotic membrane proteins, and cell culture-based membrane protein overexpression in insect cells and mammalian cells Furthermore, several chapters in this section also discuss relatively uncommon but promising strategies for expressing membrane proteins, e.g., Drosophila melanogaster, Xenopus oocytes, and Wolinella succinogenes Biochemical and functional characterization of recombinant membrane proteins is important to ensure their native-like behavior before structural studies can be undertaken Section II of Volume I encompasses several chapters that cover various methods for characterizing membrane proteins such as reconstitution in lipid environment, cross-linking, fluorescence, and spectroscopy-based approaches to investigate conformational changes and surface plasmon resonance-based strategies to study ligand–protein interactions Once the recombinant membrane protein expression has been established and functional characterization reveals native-like properties, xxiii 637 Author Index Weber, W., 215 Wedegaertner, P.B., 308–309 Wedekind, A., 173–174 Wei, J., 578 Weihofen, A., 224 Weingarten, R.A., 103–105 Weis, W.I., 332, 555–556 Weisblum, B., 11 Weiss, H.M., 146–147, 332, 409–410 Welte, T., 25 Weng, Y., 142–143 Wenger, J., 143 Westermann, B., 80 Wetzel, C., 4–5 Weyand, S., 144, 145, 553 White, J.F., 142, 192, 268, 413–414, 551–552, 553, 555–556 White, J.H., 145–146 White, W.B., 477 Whitehouse, S., 27, 31, 31f, 32, 33f, 35f, 36–37, 39, 41, 43–44, 45 Whitelaw, E., 309–310 Whiteley, E., 4–5 Whiteway, C., 406 Whiteway, M.S., 145–146 Whorton, M.R., 175, 399–401 Wickner, W., 24–25, 34, 39, 43 Wickstrom, D., 44 Wiedemann, I., 363 Wieland, F., 223 Wikstr€ om, J., 142–143 Wilden, U., 565, 587 Wildt, S., 143–144, 221 Wilkins, S., 100–101 Williams, A.J., 396 Williams, C., 394–395, 397 Willis, C.L., 268 Wilson, A., 458–459 Wilson, M.H., 309–310 Winkler, T., 287–288 Winn, M.D., 575 Winter, G., 144, 145, 553 Winzeler, E.A., 145 Wisedchaisri, G., 174–175 Wisse, L.E., 145–146 Wittelsberger, A., 529–530 Wittrup, D., 167–169 Wittrup, K.D., 167–168, 169 Wlodawer, A., 100–101 Wodak, S.J., 575–577 Wofsy, L., 534–535 Wojtowicz, W., 268–269 Wolf, M.G., 456f, 598–599 Wolynes, P.G., 600 Wong, J.P., 170 Wood, D.O., 80 Wood, D.W., 236–237 Woodbury, D., 397 Woods, R.A., 168–169, 170, 171–174 Word, J.M., 575–577 Wozniak, A., 124 Wray, V., 571–572 Wreggett, K.A., 515–516 Wriessnegger, T., 143–144 Wright, M.E., 543 Wu, B., 192, 528 Wu, C.T., 236–237 Wu, F., 394–395 Wu, H., 192, 427–428, 528 Wu, J., 268–269 Wu, L., 268–269 Wu, L.W., 578 Wu, X., 309–310 Wu, Y.L., 528–529 Wurm, F.M., 309–310 Wuu, J.J., 353–354 Wyatt, R.T., 507–508, 511–512 X Xia, Y., 427–428, 528 Xie, C., 142–143 Xie, J., 544 Xie, K., 25 Xie, X., 339 Xiong, C., 333–334 Xu, B., 268–272, 273–279 Xu, C., 394–395 Xu, D., 528–529 Xu, F., 192, 273f, 427–428, 551–552, 553, 555–556 Xu, H., 601 Xu, S.L., 103–105 Xu, T., 309–310 Xue, C.B., 528–529 638 Y Yachdav, G., 458–459 Yamada, K., 25–27 Yamamura, H., 269–270 Yamashita, A., 374–375 Yampolsky, I.V., Yan, E.C., 285, 565–566 Yan, N., 220–221 Yang, D., 192, 427–428 Yang, J.P., 360–361 Yang, K., 566, 577 Yang, X., 309–310 Yang, Z., 80 Yano, J., 192, 551–552 Yao, F., 287–288 Yao, Y., 260, 461–462 Yau, K.W., 269–270 Ye, S., 565–566 Yeagle, P.L., 406 Yee, S.W., 373–374 Yeliseev, A.A., 507–508 Yernool, D., 374–375 Yi, L., 41 Yi, M., 175 Ying, W., 285–286 York, D., 599 Young, C.L., 145, 166–178, 179 Ytterberg, A.J., 4–5 Yu, F., 476–477, 482–484 Yu, J., 142–143 Yuan, J., 44, 52 Yuchi, Z., 458–459 Yuraszeck, T., 170–171 Yurugi-Kobayashi, T., 144, 145, 151–152, 167–168, 192 Z Zabel, U., 428, 461–462, 464 Zachariae, U., 595, 597, 601 Zacharias, D.A., 459 Zaitseva, E., 285, 591–592 Zamani, M.R., 142–143 Zamboni, N., 80 Zaraisky, A.G., Zare, R.N., 175 Author Index Zeder-Lutz, G., 144, 146–147, 166–167, 553, 554 Zellnig, G., 143–144 Zemlin, F., 220 Zerangue, N., 445 Zhang, C., 192, 503–504 Zhang, G., Zhang, H., 435, 444 Zhang, J., 374, 459–460, 503–504 Zhang, L.L., 80, 598 Zhang, M., 148–150, 155–156 Zhang, R., 144, 339, 417 Zhang, T., 80 Zhang, Y., 80, 142–143 Zhang, Z., 192, 427–428, 529, 537–538 Zhao, C., 430–432, 435, 445 Zhao, J., 353 Zhao, Q., 192, 528 Zheng, Y.F., 192, 557 Zhong, X., 456–457, 458, 459 Zhong, Y., 142–143 Zhou, D., 309–313, 326 Zhou, H., 175 Zhou, Q.Y., 308–309 Zhou, X., 373–375 Zhu, R., 124 Zhu, Y., 192, 427–428 Zhuang, Y., 309–310 Zhukov, A., 500, 517, 518–519 Zhuo, R., 142–143 Ziegler, C., 373–374 Ziliox, M., 285 Ziltener, P., 340 Zimmerberg, J., 397 Zocher, M., 353, 356, 357–359, 360–361, 362–365 Zolnerciks, J.K., 413–414 Zou, Y., 142, 186–188, 427–428, 438–439, 517–518, 528, 565, 595 Zsembery, A., 260 Zulauf, M., 410–411 Z€ urn, A., 461–462, 464 Zvalova-Iooss, D., 553 Zvyaga, T.A., 590, 597 Zwicker, K., 100–101, 103–105 SUBJECT INDEX Note: Page numbers followed by “f ” indicate figures and “t ” indicate tables A ACEMBL system, 26–28, 29f, 30f, 45–46 Acriflavine resistance B (acrB) gene inactivation erythromycin sensitivity, 9, 10f FLP recombinase/FRT sites, 8–9 λ RED proteins, primer sequences, 8–9, 9t recombineering technique, Allosteric modulators, 514 Alphavirus-based expression See Semliki Forest virus (SFV) expression system Arrestin mutants characterization absorbance spectrum, 585 quantum yield determination, 585–586 receptor binding measurement, 586 Stern–Volmer analysis, 586–587 cysteine and tryptophan, 583–584 expression and purification, 570–571 labeling, 584 B Baculovirus insect cell expression system AcMNPV, 191–192 GPCR structures, 186–188, 187f protein expression large-scale agitation bioreactors, 215 small-scale protein expression, 213–214 TIPS vials, 214 WAVE bioreactors, 215–216 recombinant bacmid DNA buffer recipes, 192 cloning, 192 high-throughput technology, 205–208 PCR analysis, 204–205 Sf9, Sf21, and Hi5 cells adherent cell cultures, 190 freezing cells, 191 monitoring cell density and viability, 191 in suspension culture, 188–189, 188f, 190–191 thawing frozen stocks, 189 storage, TIPS method, 212–213 transfection, 209–210 high-throughput transfection, 211–212 Sf9 cells, 208–209, 208f small-scale transfection, 210–211 transfer vectors, 191–192, 193t Beta 2-adrenergic receptor (β2-AR), 268–269 Biacore GPCR biosensor assay protocol, 515–516 SPR for detection, 503 detergent screening, 512 instruments and specifications, 504–507, 505t Bio-Beads®, 177–178 Bovine rhodopsin HEK293S GnTI–cells cell harvesting, 322 1D4-Sepharose resin, 325 flowchart, 310–313, 312f induction-expression, 322 opsin cDNA subcloning, 313–316 PB system (see piggyBac (PB) transposon system) reconstitution and solubilization, 324 roller bottles cell culture, 320–321 spin labeling, 325 transfection process, 317–320 UV-visible spectrum, 325–326, 326f mammalian cell system, 308–309 overexpression, 308–309 C Cell-free (CF) expression systems assembled ND production, 356–357 chloroform-solubilized lipids, 356 configurations, 353–354, 355t Escherichia coli lysates, 352 639 640 Cell-free (CF) expression systems (Continued ) L-CF mode, 360–363, 362f level-I protocol development, 360 lysate preparation, 354–356 membrane protein levels, 354 MraY enzymes, 353 MraY translocases, 363–365, 364t MSP purification, 357 ND preparation, 357–359 optimization parameter, 359 purification, 365–366 stabilizing agents, 352–353 translation, improper initiation of, 359–360 workflow, 358f Cholesteryl hemisuccinate (CHS), 554–555 Cloning OsVDAC4, 57–58 pNZ8048 amplification, 89 electrocompetent, preparation of, 92 ligation and pellet paint coprecipitation, 92 preparation, 91 restriction digestion, 90, 90t, 91, 91t Contrast transfer function (CTF), 263 Cross-linking strategies chemical cross-linking DOPA-biotinylated peptides, 535 DOPA-labeled peptides, 535–537 DOPA1,Lys7(BioACA),Nle12]α-factor synthesis, 535 yeast cells expressiing ste2p, 534–535, 536f identification fragmenting cross-linked peptide–protein complex, 540–541 isolating cross-linked fragment, 541 MALDI-TOF, 541–543 tagged receptor–ligand complex, 539–540 tandem mass spectrometry, 542–543 photochemical cross-linking [Bpa1, K(BiotinACA)Nle12]α-factor cross-linking, 533–534 [Bpa1, K(BiotinACA)Nle12]α-factor synthesis, 532–533 Subject Index Bpa-labeled peptides, 533 Bpa1,Lys7(biotinylamidocaproate), Nle12]α-factor, 530 α-Fmoc[Bpa1,Nle12]α-factor synthesis, 530–532 unnatural amino acid replacement incorporation, 538–539 ligand capture, 538 Crystallization ligand-free opsin and opsin–peptide complexes, 572–573, 573f Meta II and Meta II–GαCT complex, 573–574 p44, 574 D Detergents, 252–253, 254f Dihydropyridine receptor (DHPR), 456, 456f Dodecyl maltoside (DM), 554–555 Drosophila melanogaster balancing If/CyO; Sb/TM3Ser phenotype, 229, 231 requirements, 230 chromosome mapping, 230 culture culture tubes preparation, 230 economical value, 224 requirements, 230 driver strains, 231–232 ectopic protein purification, 223 generation, 224, 225f HsSPP and DmSPP expression, 224, 236, 237f human vasopressin 1A receptor, 223 localization analysis, 231–232 membrane preparation, 232–233, 233t, 234 metabotropic glutamate receptor, 223 molecular biology, 225–227 protein purification, 234–235, 234t, 235t pUAST fly expression vector, 225, 226f rhabdomeres, 221–223, 222f Schneider cells material, 227 medium, 227, 228t transfection, 228 641 Subject Index site-specific gene insertion, 236–237 w1118 strain, 226f, 228–229 E Escherichia coli (E coli) acrB gene inactivation erythromycin sensitivity, 9, 10f FLP recombinase/FRT sites, 8–9 λ RED proteins, primer sequences, 8–9, 9t recombineering technique, antibiotic resistance markers, 7–8 green fluorescent protein, 6–7 plasmids gel-based expression analysis, 13–14, 14f LIC compatible system, 11 linker length effect, 10–11 PBAD promoter, 10 whole-cell fluorescence, 11–12 strains characterizations DNA sequencing, 15–16 in vivo functional assay, 16 plasmid copy number, 15–16 plasmid curing, 16 selection process, 14–15 transcription level, 15–16 Extra Meta II assay amount, 578, 579f applications, 580–581 disadvantages, 581–582 methodology, 578–580 phosphorylation, 582 synergism of, 588f tautomeric equilibrium, 578 F FACS See Fluorescence-activated cell sorting (FACS) FBDD See Fragment-based drug design (FBDD) Fluorescence-activated cell sorting (FACS), 80–82, 81f Fluorescent protein (FP) fusions cDNA cloning, 459 cDNA transfection, 460 cell line, 459–460 glycine-rich linkers, 459 replating, 460 results, 461 selection, 458–459 visualizing FP expression, 460 Folding monolayer approach, 70–74, 71f Fourier transform infrared (FTIR) spectroscopy binding spectra, 593–594 disadvantages, 595 meta equilibria, global fit analysis of, 591–593 preparation and measuring techniques, 589–590 R* states, monitoring of, 590 Fragment-based drug design (FBDD) GPCRs study, 507 HTS, 503–504 instruments and specifications, 504–507, 505t label-free technique, 507 FTIR See Fourier transform infrared (FTIR) Functional assays, NirC electrophysiological analysis gating process, 487–488, 487f macroscopic currents measurements, 486–487, 487f Nernst’s equation, 486–487, 487f protein:lipid ratios, 487–488 proteoliposomes and reconstitution preparation, 485–486 single-channel recordings, 486 H+ transport inside-out (everted) membrane vesicles preparation, 492 NO–2/H+ antiport activity, 492–494 SSM-based electrophysiology characterization, 489f, 490–491 interpretation, 491 proteoliposome adsorbed, 488, 489f technique and the experimental setup, 488–490 G GFP See Green fluorescent protein (GFP) GPCRs See G protein-coupled receptors (GPCRs) 642 G protein-coupled receptors (GPCRs) alphavirus-based expression (see Semliki Forest virus (SFV) expression system) baculovirus expression system (see Baculovirus insect cell expression system) crystallization approach antibody fragment-mediated approach, 551–552 cholesteryl hemisuccinate, 554–555 constructs design, 551–552 dodecyl maltoside, 554–555 focused and targeted approach, 550–551 fusion protein approach, 551–552 LCP method, 557 olfactory receptors, 558 purification strategies, 555–556 recombinant expression system, 553–554 rhodopsin expression, 550 structural genomics, 550–551 T4 lysozyme fusion, 551–552 7TMR architecture, 550 heptahelical proteins, 186 HTS of, 503–504 ICCR activation of, 426–427 alanine scanning mutagenesis approach, 427–428 β-arrestin-based assays, 428 exogenous soluble fusion domains, 427–428 functional characterization, 428 heterologous expression, 427–428 membrane protein crystallization, 427–428 microcrystallography, 427–428 receptor stabilization technologies, 427–428 smaller camelid antibodies, 427–428 soluble domain insertion, 427–428 standard GPCR functional assays, 428 in mammalian cells (see Mammalian cells, GPCR expression) membrane mimetic environments interaction, 174–175 liposomes, 177–178 Subject Index micelles, 175–177 reconstitution of β2-adrenergic receptor, 175 overexpression, 550 physiological processes, 166 plasmid design factors affecting, 168 heterologous expression of, 167–168 high copy number transformants, 169 low copy number transformants, 168–169 PCR-based methodology, 167–168 P pastoris A2AR receptor construct, 146–147, 147f cholesterol producing strain, 143–144 large-scale growth, 143 radioligand binding analysis, 148–150, 149f recombinant protein expression, 143 SMD1163 strain, 143–144 vs S cerevisiae, 146 Western blot analysis, 148–150, 149f protein activity, 174 protein expression assessment A2aR and A2bR, 170, 171f biophysical/structural characterization, 169–170 inducible promoters, 171 unfolded protein response reporter system, 170–171 yEGFP-His10, 171–173, 172f protein trafficking and localization, 173–174, 173f purification, 167 S cerevisiae confocal microscopic analysis, 153–155, 154f C-terminal GFP tagging, 145 galactose-inducible promoter, 145 in-gel fluorescence analysis, 152–153, 154f MMY strains, 145–146 recombinant protein expression, 144, 145 RFU-based expression level, 155–156, 155t vs P pastoris, 146 Subject Index yeast cell-based assay (see Yeast cell-based functional assay) solubilization, 167 SPR affinity purification method and screening, 511–512 assays sensitivity development and improvement, 512, 513f capture and reconstitution, 509–510 chemokine receptor validation, 509 detergent screening, 512 fragment screening, 517–521 light-activated receptor Rhodopsin, 508 micropatterned immobilization technique, 508 monitoring small-molecule interaction, 510–511 rhodopsin, 509 thermostabilization, 517 structural and biophysical characterization, 166–167 7-TM, 500–501 Green fluorescent protein (GFP), 6–7, 80–82, 81f GROMACS, 598–599 H HEK293S cells See Human embryonic kidney 293 suspension (HEK293S) cell system HEK293S GnTI–cells, rhodopsin expression cell harvesting, 322 1D4-Sepharose resin, 325 flowchart, 310–313, 312f induction-expression, 322 opsin cDNA subcloning, 313–316 PB system (see piggyBac (PB) transposon system) reconstitution and solubilization, 324 roller bottles cell culture, 320–321 spin labeling, 325 transfection process freezing cells, 320 mechanism, 318–319 6-well plates, 318 Western blot analysis, 319, 320f UV-visible spectrum, 325–326, 326f 643 Hi-Clamp two-electrode voltage clamp system, 260–261 High-throughput screening (HTS), 503–504 Holotranslocon (HTL) ACEMBL system, multiprotein complex, 26–28, 29f, 30f, 45–46 purification, 25–26, 28–31, 31f, 45 SecYEG-and HTL-containing PLs ATP-stimulated protein secretion, SecA ATPase, 37–39, 38f, 43 bacteriorhodopsin (BR), 32, 33, 33f Blue Native (BN)-PAGE, 32, 33–34, 34f Coomassie blue staining, 32, 33f, 36–37, 37f E coli SecY and YidC, inverted orientation of, 34, 35f membrane protein insertion activity, 41–42, 43–44 PMF-stimulated protein secretion activity, 39–41, 43 SDS-PAGE electrophoresis analysis, 32, 33f, 36–37, 37f subunit interactions, PICUP, 36–42, 37f trypsin proteolysis, 34–36, 35f HTL See Holotranslocon (HTL) HTS See High-throughput screening (HTS) Human embryonic kidney 293 suspension (HEK293S) cell system chemical transfection reagents, 286 expression plasmids, 286 gene delivery, methods of, 286 gene expression, 286 GnTI¯ cells, 285, 298–299 HEK293S-TetR cells clone selection, 275–278, 277f codon-optimized β2-AR gene, 268–269 codon-optimized TP gene, 268–269 GPCRs, purification of, 278–279 high-level inducible rod opsin gene expression, 295–298, 297f pACMV-tetO, 287–288, 288f, 294–295 pCDNA6/TR, 294 644 Human embryonic kidney 293 suspension (HEK293S) cell system (Continued ) transfection, 275–278, 277f maintenance and storage, 288–290 mammalian cell colonies, 292–293 Rho-1D4 antibody, membrane protein purification, 301–303, 302f rhodopsin, 285, 287 suspension cultures bioreactor, 300–301 calcium-free DMEM, 300 inoculation, 300 transfection stable transfection, 290–292 transient transfection, 290, 292 Human membrane proteins signal peptide peptidase, 224, 236, 237f Xenopus laevis oocytes (see Xenopus laevis oocyte system) I Immobilized metal affinity chromatography (IMAC), 357 Insect cells See Baculovirus insect cell expression system Ion channel-coupled receptor (ICCR) technology advantages, 429, 451, 451t C-terminal domains, 433–435, 434f design linker optimization, 444, 445f materials, 440 mRNA synthesis, 443–444 pGEMHE vector, 441–443, 442f T4L engineering and site-directed mutagenesis, 445–446 two-step PCR protocol, 440–441, 441f, 442t, 443t D2L-like C-ter deletion, 433–435 extrapolation, 437–439, 439f full-length receptor, 433–435 functional characterization automated TEVC protocol, 450–451 manual TEVC protocol, 449–450 mRNA microinjection, 429, 431f, 447–448 Xenopus oocytes preparation, 446–447 functional proteins, 432, 434f Subject Index GPCR activation of, 426–427 alanine scanning mutagenesis approach, 427–428 β-arrestin-based assays, 428 exogenous soluble fusion domains, 427–428 functional characterization, 428 heterologous expression, 427–428 membrane protein crystallization, 427–428 microcrystallography, 427–428 receptor stabilization technologies, 427–428 smaller camelid antibodies, 427–428 soluble domain insertion, 427–428 standard GPCR functional assays, 428 Kir3.x channels, 432, 433f, 436–437 limitations, 451, 451t M2-like C-ter deletion, 433–435 optimal expression, 429 origin, 429, 430f pharmacological properties, 435–436, 436f TEVC technique, 429–430, 431f troubleshooting, 452, 452t two-step PCR, 429, 431f Ion channels electrical recording channel conductance and gating, 400–401 current/voltage experiments, 397–398, 398f ion selectivity, 398–399 macroscopic experiments, 396–397 patch clamping, 395–396 planar lipid bilayer recording, 395, 396, 396f single-channel experiments, 397 single-channel kinetics—spontaneous closings, 401–402 liposome flux assays applications and limitations, 394–395 detection method, 391–392 extraliposomal conditions, 394 A F–efflux assay, 392–393, 392f loading liposomes, 393 A 86Rb+ uptake assay, 393 645 Subject Index unilamellar liposomes, 394 liposome reconstitution bio-beads SM2 absorption, 388 buffer preparation, 387 dialysis, 387–388 dissolve lipids, 387 freeze proteoliposomes, 388 gel filtration, 388 preformed liposomes, 390–391 purified ion-channel proteins, 387 voltage/patch-clamp techniques, 386 K KDEL receptor, 80–82 Kruskal–Wallis rank sum test, 252–253 L Lactococcus lactis analysis, 95 AT-rich codon usage, 83 buffers and media, 85–87 cloning amplification of target gene, 89, 90t electrocompetent, preparation of, 92 ligation and pellet paint coprecipitation, 92 pNZ vector preparation, 91 restriction digestion, 90, 90t, 91, 91t equipment and materials, 84–85 functional characterization, 82–83 growth of, 79, 94 high resistance, 83 HtrA, 82 inclusion bodies, 80 isolation of, 95 low transformation frequency, 83 membrane proteins, 78 nisin A promoter systems, regulation, 78, 80–82 pNZ8048, 78, 79f protocol codon optimization, 88 duration, 88 expression trials, 88f primers design, 87, 87t target protein expression, 94 transformation, 93 Large unilamellar vesicles (LUVs), 409 LCP method See Lipidic cubic phase (LCP) method Ligand-binding assays, 174 Lipidic cubic phase (LCP) method, 557 Lipodisqs bR proteolipodisqs preparation, 417–418 bR proteoliposomes preparation, 417–418 discoidal nanoscale lipid bilayer system, 416, 416f dynamic light-scattering experiment, 416–417 electron microscopy, 416–417 SMA polymers, 416–417, 416f Liposomes membrane mimetic environments, 177–178 membrane proteins reconstitution hydrophobic adsorption, 410f, 411–413 lipid preparation, 407 LUVs, 409 material size, 408 rapid dilution and dialysis, 410–411, 410f SUVs, 408 NDs preparation MSP purification, 357 preparation, 357–359 production of, 356–357 workflow, 358f recombinant transporters detergent removal, 375f, 377–378 detergent solubilization, 375f, 376–377 lipid vesicle preparation, 375–376, 375f protein incorporation, 375f, 376–377 Liposome swelling assay, 67–70, 68t, 69f M Mammalian cells, GPCR expression affinity tags, 269–270 cloning, 274 codon optimization, 268–269 flow cytometry, 274–275 gene design, 273, 273f gene synthesis, 274 materials and supplies cell lines and plasmids, 270–271 646 Mammalian cells, GPCR expression (Continued ) chemical supplies and buffers, 271–272 DMEM, 271 equipment, 272 tissue culture supplies, 270 rhodopsin N-terminus, 269–270 tetracycline inducible HEK293S cell lines (see HEK293S cells) Mann–Whitney test, 252–253 Mass spectrometry, 65–66 Matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF), 541–542 Membrane proteins reconstitution, 124–125 concentration methods, 421 Drosophila photoreceptor cells, expression in (see Drosophila melanogaster) Escherichia coli, 221 in Lactococcus lactis (see Lactococcus lactis) lipid-to-protein (L:P) ratio labeled lipid marker, 419 liposome/lipid concentration determination, 418–419 sucrose density gradient, 420–421 lipodisqs bR proteolipodisqs preparation, 417–418 bR proteoliposomes preparation, 417–418 discoidal nanoscale lipid bilayer system, 416, 416f dynamic light-scattering experiment, 416–417 electron microscopy, 416–417 SMA polymers, 416–417, 416f liposomes hydrophobic adsorption, 410f, 411–413 lipid preparation, 407 LUVs, 409 material size, 408 rapid dilution and dialysis, 410–411, 410f SUVs, 408 nanodiscs formation, 414, 414f MSP production, 414 Subject Index NTS1 reconstitution, 414–416 structure determination of, 220 Membrane scaffold proteins (MSPs) engineering of, 356–357 nanodisc (ND) technology, 352–353 purification, 357 Micelles, 175–177 Mitochondria membranes, 52 roles of, 52 Molecular dynamics (MD) simulations, Rhodopsin application of, 595 bovine rhodopsin, 596f cluster analysis, 599–600 limitations, 601 protocol, 598–599 protonation states and internal water, 597–598 receptor and complexes, preparation of, 596–597 synergism, 601 MraY See Phospho-MurNAc-pentapeptide (MraY) N Nanodiscs (ND) formation, 414, 414f MSP production, 414 NTS1 reconstitution, 414–416 O OsVDAC4 cloning, 57–58, 58f equipment, 53 expression and purification inclusion bodies, 62–65, 64f screening, 59–60, 60f soluble fraction, 60–62, 62f transformation protocol for, 58–59 functional characterization BLM, 70–74, 71f, 73f geometries, 66–67 liposome swelling assay, 67–70, 68t, 69f materials, 53–57 solutions and buffers, 55–57 tryptic digestion, 65–66 647 Subject Index P PB transposon system See piggyBac (PB) transposon system Phospho-MurNAc-pentapeptide (MraY) functional expression, requirements, 353 key role, 353 translocases activity of, 363 B subtilis and E coli, 363, 364t Photoinduced cross-linking of unmodified proteins (PICUP), 36–42, 37f Pichia pastoris, 133 A2AR receptor construct, 146–147, 147f cholesterol producing strain, 143–144 large-scale growth, 143 membrane protein preparation, 133 radioligand binding analysis, 148–150, 149f recombinant protein expression, 143 SMD1163 strain, 143–144 vs S cerevisiae, 146 Western blot analysis, 148–150, 149f piggyBac (PB) transposon system plasmids types, 310, 311f repeat-induced gene silencing, 309–310 stable mammalian cell lines, 309, 310 transposable element, 309–310 Planar bilayer lipid membrane (BLM), 70–74, 71f pNZ8048 cloning amplification, 89 electrocompetent, preparation of, 92 ligation and pellet paint coprecipitation, 92 preparation, 91 restriction digestion, 90, 90t, 91, 91t vector map of, 78, 79f Proteoliposomes (PLs) ATP-stimulated protein secretion, SecA ATPase, 37–39, 38f, 43 bacteriorhodopsin (BR), 32, 33, 33f Blue Native (BN)-PAGE, 32, 33–34, 34f Coomassie blue staining, 32, 33f, 36–37, 37f E coli SecY and YidC, inverted orientation of, 34, 35f membrane protein insertion activity, 41–42, 43–44 PMF-stimulated protein secretion activity, 39–41, 43 SDS-PAGE electrophoresis analysis, 32, 33f, 36–37, 37f subunit interactions, PICUP, 36–42, 37f trypsin proteolysis, 34–36, 35f Proton-motive force (PMF), 39–41, 43 R Radioactive uptake assay Avanti sells polycarbonate membranes, 378 background signal estimation, 381 freezing and thawing, 378 higher throughput method, 379 liquid scintillation, 381 Mini-Extruder, 378 proteoliposome solution, 378 radiolabeled substrate and valinomycin solution, 380 substrate specific activity, 381 transport reaction, 380 vacuum-filtration system, 379, 381 Whatman glass microfiber GF/B filter, 380 Radioligand binding analysis P pastoris, 148–150, 149f Y lipolytica, 135–139 Recombinant transporters radioactive uptake assay Avanti sells polycarbonate membranes, 378 background signal estimation, 381 freezing and thawing, 378 higher throughput method, 379 liquid scintillation, 381 Mini-Extruder, 378 proteoliposome solution, 378 radiolabeled substrate and valinomycin solution, 380 substrate specific activity, 381 transport behavior, 378, 379f transport reaction, 380 vacuum-filtration system, 379, 381 Whatman glass microfiber GF/B filter, 380 648 Recombinant transporters (Continued ) reconstitution process detergent removal, 375f, 377–378 detergent solubilization, 375f, 376–377 lipid vesicle preparation, 375–376, 375f protein incorporation, 375f, 376–377 Renkin equation, 70 Rhodamine-PE (Rho-PE) method, 419 Rhodopsin, 564–565 central ionic lock, 565–566 cytoplasmic ionic lock, 566 FTIR spectroscopy binding spectra, direct observation of, 593–594 disadvantages, 595 Meta equilibria, global fit analysis of, 591–593 preparation and measuring techniques, 589–590 R* states, monitoring of, 590 function of, 565–566 GPCRs, 565 MD simulations application of, 595 bovine rhodopsin, 596f cluster analysis, 599–600 limitations, 601 protocol, 598–599 protonation states and internal water, 597–598 receptor and complexes, preparation of, 596–597 synergism, 601 proteins and peptides preparations arrestin mutants, expression and purification, 570–571 heterologous expression, 569–570 isolation of rod outer segments, 567–568 ligand-free opsin preparation, 568–569 native Gt, isolation of, 568–569 phosphorylation, 569 synthesis of, 571–572 site-directed fluorescence spectroscopy applications of, 587 characteristics of, 582–583 cysteine/tryptophan arrestin mutants, 583–584 Subject Index labeling of arrestin mutants, 584 limitations, 588–589 TrIQ, 582–583 TrIQ methodology, 589 thermodynamic equilibrium, 565–566, 566s UV/VIS absorption spectroscopy (see Extra Meta II assay) X-ray crystallography (see X-ray crystallography) RoboInject system cRNA injection, 249–251, 250f, 250t needles, preparation of, 249 96-V-well plates, preparation of, 248–249 S Saccharomyces cerevisiae confocal microscopic analysis, 153–155, 154f C-terminal GFP tagging, 145 galactose-inducible promoter, 145 GPCR expression in (see G proteincoupled receptors (GPCRs)) in-gel fluorescence analysis, 152–153, 154f MMY strains, 145–146 recombinant protein expression, 144, 145 RFU-based expression level, 155–156, 155t vs P pastoris, 146 yeast cell-based assay (see Yeast cell-based functional assay) Salmonella typhimurium NirC (StNirC) electrophysiological analysis gating process, 487–488, 487f macroscopic currents measurements, 486–487, 487f Nernst’s equation, 486–487, 487f protein:lipid ratios, 487–488 proteoliposomes and reconstitution preparation, 485–486 single-channel recordings, 486 expression constructs, 478 H+ transport inside-out (everted) membrane vesicles preparation, 492 NO–2/H+ antiport activity, 492–494 isolation and reconstitution, 479–480 Subject Index overproduction, 478–479 SSM-based electrophysiology characterization, 489f, 490–491 interpretation, 491 proteoliposome adsorbed, 488, 489f technique and the experimental setup, 488–490 structural analysis crystallization, 481 diffraction data collection and processing, 481–482 FocA proteins, 480–481 structure solution and refinement, 482 three-dimensional structure, 482–484, 483f SEC See Size-exclusion chromatography (SEC) Semliki Forest virus (SFV) expression system cell cultures, 334 DNA-layered vectors, 333–334, 333f GPCR expression affinity chromatography concentration, 344 Centriprep concentration, 343 drug discovery, 339–342 examples of, 346–348, 347t hippocampal slice cultures, 342–343, 344–345 in vivo delivery, 345–346 primary neurons, 342–343, 344 SFV stocks, ultracentrifugation of, 343 structural biology, 346 recombinant SFV particle production activation of, 337 approximate titer estimations, 337–338 β-gal expression, visualization of, 338 cloning methods, 335 electroporation of RNA, 336 harvest of, 337 in vitro transcription, 335–336 RT-PCR-based titer determination, 337–338 transfection reagents, 337 replication-deficient particles, 333–334, 333f replication-proficient particles, 333–334, 333f SFV vectors, 334 649 Signal peptide peptidase (SPP), 224, 236, 237f Single particle analysis (SPA), 252, 262f Single particle reconstruction (SPR), 252, 262f CTF and phase-flipping corrections, 263 e2boxer.py program, 261 e2initialmodel, 263 iTEM software, 261–264 reference-free class averages, 263 refinement and resolution, 263 Site-directed fluorescence spectroscopy applications of, 587 characteristics of, 582–583 cysteine/tryptophan arrestin mutants, 583–584 labeling of arrestin mutants, 584 limitations, 588–589 TrIQ, 582–583, 589 Site-specific labeling methods cells expression, 469–470, 470f DHPR complex, 456, 456f FP fusions cDNA cloning, 459 cDNA transfection, 460 cell line, 459–460 glycine-rich linkers, 459 replating, 460 results, 461 selection, 458–459 visualizing FP expression, 460 poly-histidine tags Cy3/5NTA labeling procedure, 467 Cy3/5NTA purification, 466–467 Cy3/5NTA synthesis, 466 his tag cDNA cloning, 465 monitoring Cy3/5NTA labeling, 467–468 protein expression, 465 results, 468–469, 469f RyR1 cDNA, 456, 456f, 469–470, 470f structural analysis, 456–458, 457f tetracysteine (Tc) tags, biarsenicals initial labeling, 462–463 materials, 462 nonspecifically bound FlAsH/ReAsH, 463 recognition sequence, 461–462 results, 463–464, 464f 650 Size-exclusion chromatography (SEC), 554–555 Skeletal muscle membrane proteins, FRET See Site-specific labeling methods Small unilamellar vesicles (SUVs), 408 SPP See Signal peptide peptidase (SPP) SPR See Single particle reconstruction (SPR) StaRs, 516–517 Stern–Volmer analysis, 586–587 Strontium chloride (SrCl2), 252–253, 254f Styrene-maleic acid polymer (SMA), 416–417, 416f Surface plasmon resonance (SPR), 7TMRs affinity and kinetic parameters measurement, 502–503 allosteric compounds allosteric modulators, 514 allosteric molecules detection, 515–516, 515f chemokine receptor antagonists and inverse agonists, 515–516 Biacore, 503 FBDD GPCRs study, 507 HTS, 503–504 instruments and specifications, 504–507, 505t label-free technique, 507 fragment screenings confirmation of, 521–522 functional GPCRs, 517–521 thermostabilized GPCRs, 516–517 GPCRs affinity purification method and screening, 511–512 assays sensitivity development and improvement, 512, 513f capture and reconstitution, 509–510 chemokine receptor validation, 509 detergent screening, 512 functional CCR5, immobilization of, 511 light-activated receptor Rhodopsin, 508 micropatterned immobilization technique, 508 Subject Index monitoring small-molecule interaction, 510–511 rhodopsin, 509 polarized light, 501–502 sensitive and quantitative biophysical technique, 501–502 T Tandem mass spectrometry, 542–543 Tandem recombineering (TR), 26–28 Tetracycline-inducible HEK293S stable cell lines See HEK293S cells Thin layer chromatography (TLC), 363 Titerless infected-cell preservation and scale-up (TIPS), 212–213 TLC See Thin layer chromatography (TLC) Transducin, 570 Transmission electron microscopy (TEM), 242–243 TrIQ See Tryptophan-induced quenching (TrIQ) Tryptic digestion, 65–66 Tryptophan-induced quenching (TrIQ), 582–583, 583f Two-electrode voltage-clamp (TEVC) technique, 429–430 Type ryanodine receptor (RyR1), 456, 456f U UV/VIS absorption spectroscopy See Extra Meta II assay V VDAC See Voltage-dependent anion channel (VDAC) Voltage-dependent anion channel (VDAC), 52 W WAVE bioreactors, 215–216 Western blot analysis A2aR and A2bR, 170, 171f bovine rhodopsin, 319, 320f P pastoris, 148–150, 149f protein solubilization, 177–178, 179f Y lipolytica, 134–135 651 Subject Index X Xenopus laevis oocyte system buffers preparation aquatic anesthesia, 245 MBM stock solutions, 244, 245t cRNA production, 243–244 deglycosylation, 258 frog surgery, 245–246, 247f gene expression, functional and structural analysis, 242–243, 244f grid preparation, 261 Hi-Clamp two-electrode voltage clamp system, 260–261 maximal protein expression additives, 252–253 cRNA amount/time incubation, 251–252 membrane preparation and detergent screening, 253–256 membrane protein purification, 256–258, 257f negative staining, 261, 262f oocytes defolliculation, collagenase treatment, 246–248, 247f pMJB08 vector, subcloning in, 243–244 RoboInject system cRNA injection, 249–251, 250f, 250t needles, preparation of, 249 96-V-well plates, preparation of, 248–249 SPR (see Single particle reconstruction (SPR)) surface biotinylation, 259–260 TEM, 242–243 X-ray crystallography crystal contacts, 577–578 crystallization techniques cryoconditions, 574 ligand-free opsin and opsin–peptide complexes, 572–573 Meta II and Meta II–GαCT complex, 573–574 p44, 574 data collections, 574–575 electron density, 575, 576f forms of, 572 limitations, 577–578 manual rebuilding, 575–577 refinement, 575 structure solution, 575 Y Yarrowia lipolytica equipments, 125–126 flowchart analysis, 129f membrane protein preparation, 133–134 PO1d Δpah1 strain, 125 radioligand binding analysis, 135–139 small-scale expression screening, 132–133 solutions and buffers, 127–128 transformation protocol, 129–131 Western blotting analysis, 134–135 Yeast cell-based functional assay A2AR construct analysis, 158–159, 159f 3AT concentration, 157–158, 158f mechanism of, 156f Rag23 analysis, 156–157, 157f ... experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety... Thomason, L C., Costantino, N., Bubunenko, M., Datta, S., & Court, D L (2007) Recombineering: In vivo genetic engineering in E coli, S enterica, and beyond Methods in Enzymology, 421(06), 171–199... producing challenging multiprotein complexes ACEMBL affords the means to combine multiple expression elements including promoter DNAs, tags, genes Methods in Enzymology, Volume 556 ISSN 0076-6879