Methods in Protein Biochemistry (eBook)

Harald Tschesche (Herausgeber)

eBook Download: PDF

2012
378 Seiten
De Gruyter (Verlag)
978-3-11-025236-1 (ISBN)

Infos

This book presents a survey of recent developments in protein biochemistry. Top researchers in the field of protein biochemistry describe modern methods to address the challenges of protein purification by three-phase partitioning, and their folding and degradation by the functions of chaperones. The significance of peptide purity for fibril formation is addressed as well as the use of target oriented peptide arrays in palliative approaches in mucoviszidose. The design and application of protein epitope mimetics just as the structural resolving of the misfolding of various mutant proteins in serpinopathies enlarge our tools in resolving pathophysiological imbalances.

Harald Tschesche, University of Bielefeld, Germany.

Autor

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Harald Tschesche, University of Bielefeld, Germany.

Inhalt

Preface 6
Editor 8
Contents 12
List of contributing authors 8
Abbreviations 18
Acknowledgements 26
1 Three-phase partitioning 28
1.1 Method 29
1.2 The mechanism of TPP 30
1.3 A practical example – the isolation of cathepsin L from liver tissue 31
1.4 Other applications 32
2 Folding and degradation functions of molecular chaperones 40
2.1 Introduction 40
2.2 The domain structure of Hsc/Hsp70 40
2.3 The Hsc/Hsp70 reaction cycle 42
2.4 Cochaperones determine the function of Hsc/Hsp70 43
2.5 In vitro reconstitution and functional analysis of the Hsc/Hsp70 chaperone system 43
2.6 Measuring the ATPase activity of Hsc/Hsp70 45
2.7 Determining chaperone activity 45
2.8 In vitro reconstitution of chaperone-assisted ubiquitylation 46
2.9 Concluding remarks 48
3 Membrane protein folding in detergents 50
3.1 Introduction 50
3.2 Interactions of membrane proteins with detergents 51
3.3 Techniques to characterize TM proteins in detergents 56
3.4 Applications of TM protein-detergent complexes 62
3.5 Conclusions 68
4 Glycoprotein-folding quality control in the endoplasmic reticulum 74
4.1 Introduction 74
4.2 Glycoprotein-folding quality control (QC) 74
4.3 The UGGT 76
4.4 GII 79
4.5 CNX and CRT 81
4.6 ERp57 84
4.7 Methods to study glycoprotein folding QC 85
5 Conformational dynamics in peptides and proteins studied by triplet-triplet energy transfer 100
5.1 Introduction 100
5.2 Concept of TTET experiments to study intrachain loop formation in polypeptide chains 100
5.3 Diffusion-controlled loop formation in unstructured polypeptide chains 106
5.4 Detection of fast conformational fluctuations in folded peptides and proteins by TTET 112
5.5 Conclusions 118
6 Protein import into the intermembrane space of mitochondria 122
6.1 Introduction 122
6.2 The mitochondrial IMS 122
6.3 The mitochondrial disulfide relay 123
6.4 The sulfhydryl oxidase Erv1 123
6.5 The oxidoreductase Mia40 125
6.6 Substrates of the mitochondrial disulfide relay 125
6.7 Methods to study mitochondrial protein translocation 126
6.8 General comments to the analysis of thiol-disulfide redox states 132
6.9 Outlook 134
7 On-membrane identification of gel-resolved proteins by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) 140
7.1 Introduction 140
7.2 Methods for identifying proteins electroblotted onto the PVDF membrane 143
7.3 General comments to the analysis of proteins on membranes 145
7.4 PVDF membranes or diamond-like carbon-coated (DLC) stainless steel plates? 151
7.5 Concluding remarks 151
8 Analysis of protein complexes using chemical cross-linking and mass spectrometry 154
8.1 Introduction 154
8.2 Reagents for chemical cross-linking 154
8.3 The chemical cross-linking workflow 158
8.4 MS and data analysis 160
8.5 Practical examples 163
8.6 The use of spatial constraints for modeling 164
8.7 Conclusion and outlook 165
9 Single-crystal spectroscopy correlated with X-ray crystallography provides complementary perspectives on macromolecular function 170
9.1 Introduction 170
9.2 Ionizing radiation: essential for crystal structures a problem and a reagent173
9.3 Cofactors in biology provide spectroscopic access to reaction cycles 174
9.4 Single-crystal spectroscopy correlated with X-ray diffraction 177
9.5 Correlated studies at beamline X26-C of the NSLS 180
9.6 Future prospects 186
10 Wide-angle X-ray solution scattering (WAXS) 192
10.1 Introduction 192
10.2 Sample preparation 193
10.3 Sample-handling robot 194
10.4 Data collection 195
10.5 Data processing 196
10.6 Structural information 198
10.7 Size and shape 198
10.8 Secondary and tertiary structure 199
10.9 Quaternary structure 200
10.10 Structural changes 202
10.11 Unfolding 204
10.12 Molecular modeling 206
10.13 Modeling of structural fluctuations 207
10.14 Outlook 209
11 Where purity matters: recombinant versus synthetic peptides in beta amyloid formation 214
11.1 Amyloid fibrils possess a defined quaternary structure 214
11.2 The importance of purity for reproducible kinetics of amyloid fibril formation in vitro: the Aß as an example 216
11.3 Future challenges for the characterization of fibrillar structures 219
12 Chemical modification of proteins in living cells 224
12.1 Introduction 224
12.2 Site-specific labeling of proteins with chemical probes 225
12.3 Selecting an appropriate labeling technique 233
12.4 Live cell applications 233
12.5 Technical Protocols for SNAP- tag labeling 238
13 Proteomics of human bronchoalveolar lavage fluid: discovery of biomarkers of chronic obstructive pulmonary disease (COPD) with difference gel electrophoresis (DIGE) and mass spectrometry (MS) 246
13.1 Introduction 246
13.2 Application of DIGE platform to COPD biomarker discovery 249
13.3 Outlook 260
14 Proteomic analysis of Duchenne muscular dystrophy (DMD) 262
14.1 Introduction 262
14.2 Materials 263
14.3 Methods 264
14.4 Results and Discussion 267
14.5 Conclusion 275
15 Target-oriented peptide arrays in a palliative approach to cystic fibrosis (CF) 276
15.1 Introduction 276
15.2 PDZ domains 277
15.3 CF 279
15.4 Role of PDZ domains in CFTR trafficking 280
15.5 Target-oriented peptide arrays 281
15.6 An engineered peptide inhibitor of CAL extends the half-life of AF508-CFTR 283
15.7 Methods 287
15.8 Outlook 291
16 Probing protein dynamics in vivo using backbone cyclization: bacterial acyl carrier protein as a case study 298
16.1 Introduction 298
16.2 In vivo protein cyclization, biophysical analyses and functional assays 302
16.3 Outlook 315
17 The protein epitope mimetic approach to protein-protein interaction inhibitors 322
17.1 Introduction 322
17.2 Mechanisms of protein-protein interactions 322
17.3 Small-molecule screening approaches 326
17.4 Protein epitope mimetic approaches 328
18 The structural biology of a1-antitrypsin deficiency and the serpinopathies 352
18.1 Clinical phenotypes of the serpinopathies 352
18.2 The serpin mechanism of protease inhibition 354
18.3 Folding, misfolding and polymerization 355
18.4 Serpin folding 357
18.5 Dissecting the pathways of polymerization 357
18.6 Cellular processing of polymers 364
18.7 Stem cell technology to generate models of disease 367
18.8 Conclusions 367
Index 372

Produkt-Details

• Erscheint lt. Verlag: 23.12.2012
• Zusatzinfo: 150 col. ill.
• Verlagsort: Berlin/Boston
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Technik
• Schlagworte: Analytical Methods • Analytische Methoden • Biochemie • biochemistry • Proteine • proteins
• ISBN-10: 3-11-025236-8 / 3110252368
• ISBN-13: 978-3-11-025236-1 / 9783110252361