fluorescence quenching by FAPol fluo , 397, 399
fluorescence spectroscopy, 397–400
formation, 238–266
FRET with FAPol fluo , 397, 399
functionality following immobilization using
BAPol, 597
immobilization using BAPol, 595, 596, 610
immobilization using HistAPol, 608–610
immobilization using ImidAPol, 612
immobilization using OligAPol, 613, 614
infra-red and Raman spectroscopy, 394–397
intermolecular contacts, 283–286
mapped by heteronuclear dipole-to-dipole
cross-relaxation, 470
mapped by MD, 277
mapped by NMR, 276, 469, 470
lipid binding, 275
lipid composition of MP/lipid/SMA complexes
(SMALPs), 277, 278
MALDI-TOF analyses, 633–636
MD simulations, 284, 303
methods of formation, 250
MP functionality, 296–305
MP/NVoy complexes, 282
MP stability, 296–305
NMR data
solution NMR studies of APol-trapped MPs, 462
OligAPol-mediated immobilization
with multiplexing, 615
optical spectroscopy, 382
overview, 238, 249
of composition, 273
of optical spectroscopy studies, 390
of publications, 247
of solution studies, 409, 439
of studies of their composition, structure and
solution properties, 272
particle size and organization, 279–282
protocol for APol-mediated immobilization on SPR
chips (Protocol 13.1), 617–619
protocol for AUC analysis (Protocol 9.1), 440–443
protocol for crystallizing ternary MP/APol/detergent
complexes (Protocol 11.1), 523–524
protocol for ESI-IM-MS analysis (Protocol 14.1),
648–654
protocol for ESI-MS analysis (Protocol 14.1), 648–654
protocol for preparing SP cryo-EM samples (Protocol
12.1), 579–583
protocol for SEC analysis (Protocol 9.2), 444–446
protocols for determining the mass of bound APol
(Protocol 5.2), 314–318
recognition by antibodies following immobilization
using BAPol, 595, 597, 601, 602
recognition by antibodies following immobilization
using HistAPol, 610, 611
recognition by antibodies following immobilization
using OligAPol, 614
residence time of A8-35 octyl chains at the surface
of OmpX, 304
reversibility of the aggregation induced by removing
free APols, 418, 419
SANS studies, 432–436
SAXS studies, 437
SEC analysis, 417–419, 439, 444–446
SGSV comparison of LHCII/A8-35 and LHCII/NAPol
complexes, 414, 416
studies by AUC, overview, 406, 409, 439
studies by BN-PAGE, overview, 406, 409
studies by DLS, overview, 406, 409, 439
studies by SANS, overview, 406, 409, 439
studies by SAXS, overview, 406, 409, 439
studies by SEC, overview, 406, 409, 439
studies by SGSV, overview, 406, 409
SV-AUC analysis, 425, 427
topological and conformational studies using labeled
APols, 401
transfer to lipid black films, 348
transfer to lipidic mesophases, 310
transfer to nanodiscs, 319–322
transfer to other environments, 306–310
use of FAPols in AUC experiments, 425, 427
(see also Amphipol-trapped MPs)
Membrane protein crystallization
choice of detergent, 505
in detergent solutions, 503–506
engineering target MPs, 500
formation and organization of MP crystals, 500–508
insertion of water-soluble domains, 500, 501
in lipidic mesophases (in meso), 506–509
organization of detergent in Type II crystals, 503
overview, 505
overview of resolution achieved, 504
phase separation, 504
screening of orthologs, 500
Type I crystals, 501
Type II crystals, 501, 502
use of small amphiphiles, 505
use of water-soluble binders, 500, 502
Membrane protein/detergent complexes
detergent substituting to lipids, 69, 70
making detergents less aggressive, 81–83
making MPs more resistant, 84–87
MD simulations, 69
MP instability, 70–72
visualized by neutron diffraction, 68, 69
visualized by X-ray diffraction, 69
Membrane protein/lipid interactions
bound lipids, 25–31
evolutionary conservation of lipid binding sites, 30
fluid mosaic model, 22–31
Membrane protein/NVoy complexes
SEC studies, 420
Membrane protein overexpression
in vitro, by cell-free expression (CFE), 334
in vivo, as inclusion bodies, 334
in vivo, under a functional form, 333
Membrane proteins
biosynthesis, 48–50
Index
699
fluorescence spectroscopy, 397–400
formation, 238–266
FRET with FAPol fluo , 397, 399
functionality following immobilization using
BAPol, 597
immobilization using BAPol, 595, 596, 610
immobilization using HistAPol, 608–610
immobilization using ImidAPol, 612
immobilization using OligAPol, 613, 614
infra-red and Raman spectroscopy, 394–397
intermolecular contacts, 283–286
mapped by heteronuclear dipole-to-dipole
cross-relaxation, 470
mapped by MD, 277
mapped by NMR, 276, 469, 470
lipid binding, 275
lipid composition of MP/lipid/SMA complexes
(SMALPs), 277, 278
MALDI-TOF analyses, 633–636
MD simulations, 284, 303
methods of formation, 250
MP functionality, 296–305
MP/NVoy complexes, 282
MP stability, 296–305
NMR data
solution NMR studies of APol-trapped MPs, 462
OligAPol-mediated immobilization
with multiplexing, 615
optical spectroscopy, 382
overview, 238, 249
of composition, 273
of optical spectroscopy studies, 390
of publications, 247
of solution studies, 409, 439
of studies of their composition, structure and
solution properties, 272
particle size and organization, 279–282
protocol for APol-mediated immobilization on SPR
chips (Protocol 13.1), 617–619
protocol for AUC analysis (Protocol 9.1), 440–443
protocol for crystallizing ternary MP/APol/detergent
complexes (Protocol 11.1), 523–524
protocol for ESI-IM-MS analysis (Protocol 14.1),
648–654
protocol for ESI-MS analysis (Protocol 14.1), 648–654
protocol for preparing SP cryo-EM samples (Protocol
12.1), 579–583
protocol for SEC analysis (Protocol 9.2), 444–446
protocols for determining the mass of bound APol
(Protocol 5.2), 314–318
recognition by antibodies following immobilization
using BAPol, 595, 597, 601, 602
recognition by antibodies following immobilization
using HistAPol, 610, 611
recognition by antibodies following immobilization
using OligAPol, 614
residence time of A8-35 octyl chains at the surface
of OmpX, 304
reversibility of the aggregation induced by removing
free APols, 418, 419
SANS studies, 432–436
SAXS studies, 437
SEC analysis, 417–419, 439, 444–446
SGSV comparison of LHCII/A8-35 and LHCII/NAPol
complexes, 414, 416
studies by AUC, overview, 406, 409, 439
studies by BN-PAGE, overview, 406, 409
studies by DLS, overview, 406, 409, 439
studies by SANS, overview, 406, 409, 439
studies by SAXS, overview, 406, 409, 439
studies by SEC, overview, 406, 409, 439
studies by SGSV, overview, 406, 409
SV-AUC analysis, 425, 427
topological and conformational studies using labeled
APols, 401
transfer to lipid black films, 348
transfer to lipidic mesophases, 310
transfer to nanodiscs, 319–322
transfer to other environments, 306–310
use of FAPols in AUC experiments, 425, 427
(see also Amphipol-trapped MPs)
Membrane protein crystallization
choice of detergent, 505
in detergent solutions, 503–506
engineering target MPs, 500
formation and organization of MP crystals, 500–508
insertion of water-soluble domains, 500, 501
in lipidic mesophases (in meso), 506–509
organization of detergent in Type II crystals, 503
overview, 505
overview of resolution achieved, 504
phase separation, 504
screening of orthologs, 500
Type I crystals, 501
Type II crystals, 501, 502
use of small amphiphiles, 505
use of water-soluble binders, 500, 502
Membrane protein/detergent complexes
detergent substituting to lipids, 69, 70
making detergents less aggressive, 81–83
making MPs more resistant, 84–87
MD simulations, 69
MP instability, 70–72
visualized by neutron diffraction, 68, 69
visualized by X-ray diffraction, 69
Membrane protein/lipid interactions
bound lipids, 25–31
evolutionary conservation of lipid binding sites, 30
fluid mosaic model, 22–31
Membrane protein/NVoy complexes
SEC studies, 420
Membrane protein overexpression
in vitro, by cell-free expression (CFE), 334
in vivo, as inclusion bodies, 334
in vivo, under a functional form, 333
Membrane proteins
biosynthesis, 48–50
Index
699