Methods in Protein Biochemistry

Methods in Protein Biochemistry......Page 4
Preface......Page 6
List of contributing authors......Page 8
Contents......Page 12
Abbreviations......Page 18
Acknowledgements......Page 26
1 Three-phase partitioning......Page 28
1.1 Method......Page 29
1.2 The mechanism of TPP......Page 30
1.3 A practical example – the isolation of cathepsin L from liver tissue......Page 31
1.4.3 Enhanced activity......Page 32
1.4.5 Denaturation of hemoglobin......Page 34
References......Page 35
2.2 The domain structure of Hsc/Hsp70......Page 40
2.3 The Hsc/Hsp70 reaction cycle......Page 42
2.5.1 Purification of Hsc70......Page 43
2.5.2 Purification of Hsp40......Page 44
2.7 Determining chaperone activity......Page 45
2.8.1 Purification of the E1 ubiquitin-activating enzyme......Page 46
2.8.4 Recombinant expression of Raf-1......Page 47
References......Page 48
3.1 Introduction......Page 50
3.2.2 Detergent properties......Page 51
3.2.4 Overview of commonly used detergents for membrane protein characterization......Page 52
3.2.4.1 Anionic – SDS......Page 53
3.2.4.2 Zwitterionic – DPC......Page 54
3.2.4.5 Other detergents......Page 55
3.3.1 General considerations......Page 56
3.3.2.2 Fourier transform infrared spectroscopy (FTIR)......Page 57
3.3.3.1 Size exclusion chromatography (SEC)......Page 58
3.3.3.3 Determination of helix-helix association of TM segments in detergents by FRET: experimental procedures......Page 59
3.3.4.1 X-ray crystallography......Page 61
3.4.1 Comparing detergent-solubilized states with membrane proteins in lipid bilayers......Page 62
3.4.1.2 Designed TM segments (AI series)......Page 64
3.4.2.2 Cystic fibrosis transmembrane regulator (CFTR)......Page 66
References......Page 68
4.2 Glycoprotein-folding quality control (QC)......Page 74
4.3 The UGGT......Page 76
4.3.1 Substrate recognition by UGGT......Page 77
4.4 GII......Page 79
4.5.1 CRT and CNX structures......Page 81
4.5.2 Ligand binding to CRT and CNX......Page 82
4.6 ERp57......Page 84
4.7.1 Assay for UGGT......Page 85
4.7.3 Purifi cation of GII and UGGT from rat liver......Page 86
4.7.4 Indirect analysis of N-glycans present in 35S-labeled glycoproteins......Page 88
4.7.5.1 Labeling of N-glycans......Page 89
4.7.5.2 Extraction of protein-bound N-glycans......Page 90
References......Page 91
5.2 Concept of TTET experiments to study intrachain loop formation in polypeptide chains......Page 100
5.2.1 Kinetics of TTET coupled to polypeptide chain dynamics......Page 102
5.2.2 Test for suitability of a donor-acceptor pair to directly measure chain dynamics......Page 103
5.2.3 Practical aspects of TTET measurements......Page 105
5.3.1 Kinetics of intrachain diffusion......Page 106
5.3.2 Effect of loop size on the dynamics in flexible polypeptide chains......Page 107
5.3.3 Effect of amino acid sequence on chain dynamics......Page 109
5.3.4 Chain diffusion in natural protein sequences......Page 110
5.3.6 The speed limit for protein folding......Page 111
5.4.1 Kinetic model of TTET coupled to a conformational equilibrium......Page 112
5.4.3 Dynamics in α-helical peptides......Page 114
5.4.4 Fluctuations in the native and unfolded state of the villin headpiece subdomain (HP35)......Page 116
5.5 Conclusions......Page 118
References......Page 119
6.2 The mitochondrial IMS......Page 122
6.4 The sulfhydryl oxidase Erv1......Page 123
6.6 Substrates of the mitochondrial disulfide relay......Page 125
6.7.1 Growth of yeast cells......Page 126
6.7.3 Synthesis of radiolabeled proteins......Page 127
6.7.5 Purification of the components of the mitochondrial disulfide relay......Page 128
6.7.6 In vitro reconstitution of the mitochondrial disulfide relay with purified proteins......Page 130
6.8.1 Stop of thiol-disulfide exchange......Page 132
6.8.3 Reduction of disulfides......Page 133
6.9 Outlook......Page 134
References......Page 135
7.1 Introduction......Page 140
7.2 Methods for identifying proteins electroblotted onto the PVDF membrane......Page 143
7.3.1 Advantages of gel electrophoresis......Page 145
7.3.2 Efficiency of electroblotting......Page 146
7.3.3 Efficiency of mass spectrometric analysis......Page 147
7.3.4 Large scale of mass spectrometric analysis......Page 149
References......Page 151
8.2 Reagents for chemical cross-linking......Page 154
8.2.1 Conventional homobifunctional cross-linking reagents......Page 155
8.2.2 Functionalized cross-linking reagents and associated workflows......Page 156
8.3 The chemical cross-linking workflow......Page 158
8.4.1 Mass spectrometric analysis of cross-linked samples......Page 160
8.4.2 Data analysis......Page 161
8.5.2 The 26S proteasome......Page 163
8.6 The use of spatial constraints for modeling......Page 164
8.7 Conclusion and outlook......Page 165
References......Page 166
9.1 Introduction......Page 170
9.2 Ionizing radiation: essential for crystal structures; a problem and a reagent......Page 173
9.3 Cofactors in biology provide spectroscopic access to reaction cycles......Page 174
9.4 Single-crystal spectroscopy correlated with X-ray diffraction......Page 177
9.5 Correlated studies at beamline X26-C of the NSLS......Page 180
9.5.1 An example of correlated studies of an FAD-dependent system......Page 182
9.5.2 An example of correlated studies from a metalloenzyme......Page 184
9.6 Future prospects......Page 186
References......Page 187
10.1 Introduction......Page 192
10.2 Sample preparation......Page 193
10.3 Sample-handling robot......Page 194
10.4 Data collection......Page 195
10.5 Data processing......Page 196
10.7 Size and shape......Page 198
10.8 Secondary and tertiary structure......Page 199
10.9 Quaternary structure......Page 200
10.10 Structural changes......Page 202
10.11 Unfolding......Page 204
10.12 Molecular modeling......Page 206
10.13 Modeling of structural fl uctuations......Page 207
10.14 Outlook......Page 209
References......Page 210
11.1 Amyloid fibrils possess a defined quaternary structure......Page 214
11.2 The importance of purity for reproducible kinetics of amyloid fibril formation in vitro: the Aβ as an example......Page 216
11.3 Future challenges for the characterization of fibrillar structures......Page 219
References......Page 221
12.1 Introduction......Page 224
12.2.1 Self-labeling proteins and peptides......Page 225
12.2.1.1 Tetracysteine and tetraserine tags......Page 226
12.2.1.2 SNAP- and CLIP-tags......Page 227
12.2.1.3 HaloTag......Page 228
12.2.2 Enzyme-mediated labeling......Page 229
12.2.2.2 Biotin and lipoic acid ligases......Page 230
12.2.3 Summary......Page 231
12.4.1 Pulse-chase labeling......Page 233
12.4.2 Superresolution microscopy......Page 236
12.4.4 Other Applications......Page 237
12.5.1 Standard protocol for labeling in living cells......Page 238
12.5.2 Technical notes......Page 239
References......Page 241
13.1.1 Proteomics and biomarker discovery......Page 246
13.1.2 COPD......Page 247
13.1.4 DIGE......Page 248
13.2 Application of DIGE platform to COPD biomarker discovery......Page 249
13.2.1 Processing of BAL samples......Page 250
13.2.3 Two-dimensional gel electrophoresis......Page 252
13.2.4 Image acquisition......Page 254
13.2.5 Differential expression analysis......Page 255
13.2.6 Protein identifi cation......Page 256
References......Page 260
14.1 Introduction......Page 262
14.2 Materials......Page 263
14.3.2 Protein extraction......Page 264
14.3.5 Staining of proteins......Page 265
14.3.9 MALDI MS......Page 266
14.4.1 Muscle cell culture......Page 267
14.4.2 Two-dimensional gel electrophoresis......Page 268
14.4.2.2 MS......Page 269
14.4.2.3 Identified proteins......Page 271
References......Page 275
15.1 Introduction......Page 276
15.2 PDZ domains......Page 277
15.3 CF......Page 279
15.4 Role of PDZ domains in CFTR trafficking......Page 280
15.5 Target-oriented peptide arrays......Page 281
15.6 An engineered peptide inhibitor of CAL extends the half-life of ΔF508-CFTR......Page 283
15.7.1 PDZ domain and peptide motif nomenclature......Page 287
15.7.2 Improved method of inverted peptides......Page 288
15.7.5 Binding studies of cellulose-bound peptides......Page 289
15.7.8 Ussing chamber experiments......Page 290
References......Page 291
16.1.1 Studying protein structures and dynamics in living cells......Page 298
16.1.2.1 Cyclic peptides and proteins found in nature......Page 299
16.1.2.2 Engineering artificial cyclic peptides and proteins......Page 300
16.1.3 Motivation for the presented methodology......Page 301
16.2.1 Plasmid construction......Page 302
16.2.2 In vivo protein cyclization......Page 304
16.2.3.1 Purification......Page 307
16.2.3.2 CD spectroscopy......Page 308
16.2.3.3 Steady-state tryptophan fluorescence spectroscopy......Page 309
16.2.3.4 Charge state distribution......Page 311
16.2.4.1 Engineering an E. coli ACP-knockout strain......Page 312
16.2.4.3 In vivo growth complementation assay......Page 314
16.3 Outlook......Page 315
References......Page 316
17.2 Mechanisms of protein-protein interactions......Page 322
17.3 Small-molecule screening approaches......Page 326
17.4 Protein epitope mimetic approaches......Page 328
17.4.1 Helix mimetics......Page 329
17.4.2 ß-Hairpin mimetics......Page 334
References......Page 340
18.1.2 Familial encephalopathy with neuroserpin inclusion bodies (FENIB)......Page 352
18.2 The serpin mechanism of protease inhibition......Page 354
18.3.1 Polymer linkage via β-sheet A......Page 355
18.3.2 Alternative pathways to polymerization......Page 356
18.5.1 Limited proteolysis and hydrogen/deuterium exchange......Page 357
18.5.3 Mass spectrometry......Page 358
18.5.3.1 Structural analysis of the polymer by ion mobility spectrometry......Page 360
18.5.4 Two-color coincidence detection (TCCD)......Page 362
18.6 Cellular processing of polymers......Page 364
References......Page 367
Index......Page 372