Isotope Labeling of Biomolecules - Applications

 
 
Academic Press
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  • erschienen am 11. Januar 2016
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  • 482 Seiten
 
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978-0-12-803074-5 (ISBN)
 
Isotope Labeling of Biomolecules: Applications, the latest in the Methods in Enzymology series, focuses on stable isotope labeling methods and applications for biomolecules. This practical guide to biomolecular labeling looks at new techniques that are becoming widely used.
  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Focuses on stable isotope labeling of biomolecules, which is important for structural studies of proteins and nucleic acids
0076-6879
  • Englisch
  • San Diego
  • |
  • USA
Elsevier Science
  • 32,08 MB
978-0-12-803074-5 (9780128030745)
0128030747 (0128030747)
weitere Ausgaben werden ermittelt
  • Front Cover
  • Isotope Labeling of Biomolecules - Applications
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Section I: Nuclear Magnetic Resonance Spectroscopy
  • Chapter One: Application of Natural Isotopic Abundance 1H-13C- and 1H-15N-Correlated Two-Dimensional NMR for Evaluation o ...
  • 1. Introduction
  • 2. Preparation of Protein Therapeutic Samples
  • 2.1. Considerations for Sample Conditions
  • 2.2. Sample Preparation of the Intact NISTmAb
  • 2.3. Enzymatic Digest of mAbs into Fab and Fc Domains
  • 2.4. Sample Preparation of Filgrastim
  • 3. Acquisition of 2D Spectra
  • 3.1. Amide 1H-15N-Correlated Spectra
  • 3.2. Methyl 1H-13C-Correlated Spectra
  • 3.3. Nonuniform Sampling Method
  • 4. Processing 2D-Correlated Spectra
  • 4.1. Processing Uniformly Sampled Data
  • 4.2. Processing Nonuniformly Sampled Data
  • 5. Comparative Analysis of 2D-Correlated Spectra
  • 6. Perspectives
  • Acknowledgments
  • References
  • Chapter Two: A Semiautomated Assignment Protocol for Methyl Group Side Chains in Large Proteins
  • 1. Introduction
  • 2. Labeling of Side Chain Methyl Groups for Large Proteins
  • 3. Methyl Labeling Protocol for the cAMP-Dependent Protein Kinase A
  • 3.1. Materials
  • 3.2. Purification of PKA-C
  • 3.3. Materials
  • 4. Semiautomated Methyl-Group Resonance Assignment Strategies
  • 5. Semiautomated Assignment Protocol Using FLAMEnGO 2.0
  • 5.1. Materials
  • 5.2. System Requirements and Specifications
  • 5.3. Input File Formats
  • 5.3.1. Initial Random Assignment
  • 5.3.2. 3D C,C,H 13C-HMQC-NOESY-13C-HMQC
  • 5.3.3. [1H,13C]-HMQC/15N Amide
  • 5.3.4. Paramagnetic Relaxation Enhancement Data
  • 5.3.5. Prediction of Methyl Group Chemical Shifts from the PDB File
  • 5.3.6. Residue Type
  • 5.4. Instructions to Run FLAMEnGO 2.0 GUI
  • 5.5. Output Files
  • 5.5.1. Score and Assignment
  • 5.5.2. Summary
  • 6. Conclusions and Perspectives
  • Acknowledgments
  • References
  • Chapter Three: 19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR
  • 1. Introduction
  • 2. General Enzymatic Synthesis of 2F-ATP, 5F-CTP, and 5F-UTP
  • 3. Stability of Modified RNA
  • 3.1. Impact of Fluorine Substitutions on Base Pairing
  • 3.2. Thermal Stability of Fluoro-Modified RNA
  • 4. Diagnostics of 19F-Chemical Shifts
  • 5. NMR-Assignment Strategies for Fluoro-Modified RNA
  • 5.1. General NMR Hardware Considerations
  • 5.2. 19F,19F-NOESY
  • 5.3. 19F,1H-HOESY
  • 5.4. 1H,19F-HMBC
  • 6. Coupling Measurements in Fluoro-Modified RNA
  • 6.1. Scalar J-Coupling Measurements
  • 6.2. RDC Measurements
  • 7. Solvent-Induced Isotope Shifts
  • 8. Ligand-Induced Chemical Shift Changes
  • 9. Conclusion and Remarks
  • Acknowledgments
  • References
  • Chapter Four: Applying Thymine Isostere 2,4-Difluoro-5-Methylbenzene as a NMR Assignment Tool and Probe of Homopyrimidine...
  • 1. Introduction
  • 2. Preparation of dF-Substituted Oligonucleotides for NMR Analysis
  • 2.1. Design of Oligonucleotides
  • 2.2. Reagents
  • 2.3. Synthesis of dF-Substituted Oligonucleotides
  • 2.3.1. Purification of dF-Substituted Oligonucleotides
  • 2.3.2. Sample Conditioning for NMR Spectroscopy
  • 2.3.3. Exchange of Samples into 99.96% D2O
  • 3. NMR Methods
  • 3.1. Proton NMR Data Acquired in 90%:10% H2O:D2O
  • 3.2. Proton and Fluorine NMR Data Acquired in 99.96% D2O
  • 4. dF as an NMR Assignment Tool
  • 4.1. Illustration of the 1H Imino Resonance Assignment Strategy Using dF Substitution
  • 4.2. Illustration of the 1H Nonexchangeable Resonance Assignment Strategy Using dF Substitution
  • 4.3. Quantifying Chemical Shift Perturbations in the dF-Modified Constructs
  • 4.3.1. Computation of Chemical Shift Differences
  • 4.3.2. Visualization of Chemical Shift Differences for All Constructs
  • 5. Structural and Dynamic Perturbations Observed by dF Substitution
  • 5.1. Altered Sugar Pucker Conformations in the PPT dF Series
  • 5.2. Changes in the Dynamic Equilibrium of the TATA Box DNA Duplex
  • 5.3. An Orthogonal Gel Assay Correlating dF NMR Results from TATA Box DNA with TATA Box Protein Binding
  • 5.3.1. TBP-Binding Assay
  • 5.3.2. Binding of TBP to Unmodified and dF-Substituted TATA Box DNA Duplexes
  • 5.3.3. Relative Thermodynamics and Kinetics of TBP:DNA Complexes
  • 6. Conclusions
  • Acknowledgments
  • References
  • Section II: Scattering Techniques
  • Chapter Five: Biomolecular Deuteration for Neutron Structural Biology and Dynamics
  • 1. The Motivation for Macromolecular Deuteration Approaches
  • 1.1. Biological Small-Angle Neutron Scattering
  • 1.2. Medium and High Resolution: Protein Crystallography and Fiber Diffraction
  • 1.3. Neutron Reflection
  • 1.4. Dynamics
  • 2. Concepts of In Vivo Deuteration
  • 2.1. Expression of Deuterated Macromolecules in the Prokaryote E. coli
  • 2.1.1. Adaptation to Deuterium-Containing Media
  • 2.1.2. Choice of Media and Culture Conditions
  • 2.1.3. Large-Scale Preparation of Fully Deuterated Cell Components
  • 2.1.4. Large-Scale Preparation of Fully Deuterated DNA
  • 2.1.5. In Vivo Deuteration of Transfer RNA
  • 2.1.6. Expression of Deuterated Recombinant Proteins
  • 2.1.6.1. A General Protocol for Perdeuteration of Recombinant Proteins Using High Cell Density Cultures, Minimal Medium, ...
  • 2.1.6.2. Matchout Protein Deuteration in High Cell Density Cultures
  • 2.1.6.3. Alternative Protocols for Deuteration of Recombinant Proteins
  • 2.1.6.4. Specific Labeling Approaches for the Study of Membrane Proteins by SANS
  • 2.2. Expression of Deuterated Macromolecules in the Eukaryote P. pastoris
  • 2.2.1. The P. pastoris Expression System
  • 2.2.2. Perdeuterated Lipids
  • 2.2.3. Perdeuteration of Proteins in P. pastoris Using Minimal Growth Medium
  • 2.3. Alternative Expression Systems
  • 2.3.1. Cyanobacterial Expression Systems
  • 2.3.2. Deuteration in Halobacteria
  • 2.3.3. Baculovirus and Mammalian Cell Expression Systems
  • 2.3.4. Cell-Free Synthesis
  • 3. Levels of Specificity in Deuterium Labeling
  • 3.1. Perdeuteration
  • 3.2. Random Fractional Deuteration (Partial Deuteration)
  • 3.3. Amino Acid Type-Specific Labeling
  • 3.4. Segmental Isotope Labeling
  • 3.5. Deuterated Peptides
  • 4. Conclusion and Perspective
  • Acknowledgments
  • References
  • Chapter Six: Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures ...
  • 1. Introduction
  • 2. SANS from Biological Molecules in Solution
  • 2.1. Contrast and Scattering Intensity
  • 2.2. Radius of Gyration and Forward Scattering Intensity
  • 2.3. Contrast Variation
  • 2.4. Contrast Match Point Analysis of the SANS Contrast Variation Data
  • 2.5. Separation of the Radii of Gyration in a Two-Component Complex
  • 2.6. Separating the Scattering Intensities in a Two-Component Complex
  • 2.7. Structure Modeling
  • 3. Materials and Methods1
  • 3.1. uOMP and Skp Expression and Purification
  • 3.2. Formation of Skp-uOMP Complex
  • 3.3. SANS Data Collection
  • 3.4. Match Point and Contrast Variation Data Analysis
  • 3.5. Molecular Dynamics Simulation of Skp-OmpA Complexes
  • 3.6. Modeling of the Skp-uOMP Complexes
  • 4. Results
  • 4.1. SANS Data
  • 4.2. Match Point Analysis and Quantification of Omp Deuteration
  • 4.3. Stuhrmann and Parallel Axis Theorem Analyses
  • 4.4. Skp Structure in Solution
  • 4.5. Skp-OmpW Structure in Solution
  • 4.6. Skp-OmpA Structure in Solution
  • 5. Discussion
  • 5.1. Skp Alone in Solution
  • 5.2. Skp-uOMP Structure and Binding Mechanism
  • 5.3. Release Mechanism
  • 6. Conclusions
  • Acknowledgments
  • References
  • Chapter Seven: Deuteration in Biological Neutron Reflectometry
  • 1. Introduction
  • 1.1. Biological Neutron Reflectometry
  • 1.2. Deuteration in Biological NR
  • 2. Experimental Methods
  • 2.1. Instrumentation
  • 2.2. Deuteration of Biological Molecules
  • 2.3. Sample Requirements for Biological NR
  • 3. Data Analysis
  • 3.1. General Data Analysis
  • 3.2. Deuteration-Specific Data Analysis
  • 3.3. Uncertainty Analysis
  • 4. Conclusion
  • 5. Acknowledgments
  • References
  • Chapter Eight: Deuterium Labeling Strategies for Creating Contrast in Structure-Function Studies of Model Bacterial Outer...
  • 1. Introduction
  • 2. Brief Introduction to Neutron Reflectometry and Data Analysis
  • 3. Isolation of Deuterated LPS from Rough Strains of Escherichia coli
  • 4. Formation of Rough LPS Monolayers at the Air-Liquid Interface
  • 5. Modeling Monolayers of Rc-LPS from Neutron Reflectometry Data
  • 6. Forming Asymmetric Bilayers Using Phospholipids and Rough LPS
  • 7. Final Remarks
  • Acknowledgments
  • References
  • Chapter Nine: Essential Strategies for Revealing Nanoscale Protein Dynamics by Neutron Spin Echo Spectroscopy
  • 1. Introduction
  • 2. Essentials for Determining Nanoscale Protein Internal Motion
  • 2.1. Theory
  • 2.1.1. Nonequilibrium Statistical Mechanics and the Mobility Tensor
  • 2.2. Partial Deuteration
  • 2.3. Activation of Nanoscale Internal Motion in NHERF1: A Specific Example
  • 2.4. A Simple Four-Point Model Shows How Selective Deuteration Can Enhance the Effects of Internal Motion
  • 3. Preparation of Partially Deuterated Protein Samples for NSE Experiments
  • 3.1. Protein Expression and Purification
  • 3.2. Producing Deuterated Protein Subunit that is Contrast Matched in 100% D2O Buffer Solution
  • 3.2.1. Materials
  • 3.2.2. Procedure
  • 3.3. Reconstitution of the Partially Deuterated Protein Complex
  • 3.4. Exchanging the Proteins and Protein Complex into D2O Buffer
  • 4. Summary
  • References
  • Section III: Mass Spectrometry
  • Chapter Ten: Method for the Determination of 15N Incorporation Percentage in Labeled Peptides and Proteins
  • 1. Introduction
  • 2. Materials
  • 3. Background
  • 4. Trypsin Digestion
  • 4.1. In-Gel Digest
  • 4.2. In-Solution Digestion
  • 5. Mass Spectrometry
  • 5.1. MALDI
  • 5.2. ESI
  • 6. Program Description
  • 6.1. Program Usage
  • 7. Results
  • 8. Conclusions
  • References
  • Chapter Eleven: QconCAT: Internal Standard for Protein Quantification
  • 1. Introduction
  • 2. QconCAT Design
  • 3. Expression and Purification of QconCAT
  • 4. Characterization
  • 4.1. Purity and Molecular Weight Evaluation
  • 4.2. Determination of Stable Isotope Labeling Efficiency
  • 4.3. Concentration Determination
  • 4.4. Digestion Efficiency Evaluation
  • 4.5. Dynamic Range Determination
  • 5. Application of QconCAT
  • References
  • Chapter Twelve: Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards f...
  • 1. Introduction
  • 2. Materials and Methods
  • 2.1. Materials
  • 2.2. E. coli Strains and Plasmids
  • 2.3. Preparation of Minimal Medium
  • 2.4. DNA Procedures
  • 3. Production and Purification of E. coli 15N-Fpg Protein
  • 3.1. Cloning of fpg Into pET11a Vector
  • 3.2. Production of 15N-Fpg
  • 3.3. Purification of 15N-Fpg
  • 4. Production and Purification of 15N-hOGG1
  • 4.1. Production of 15N-hOGG1
  • 4.2. Purification of 15N-hOGG1
  • 5. Production and Purification of 15N-hNEIL1
  • 5.1. Production of 15N-hNEIL1
  • 5.2. Purification of 15N-hNEIL1
  • 6. Production and Purification of 15N-hAPE1
  • 6.1. Production of 15N-hAPE1
  • 6.2. Purification of 15N-hAPE1
  • 7. Production and Purification of 15N-hMTH1
  • 8. Mass Spectrometric Analysis of 15N-Labeled Proteins
  • 9. Applications to the Measurement of DNA Repair Proteins
  • 9.1. Development of Methodologies
  • 9.2. Measurement of DNA Repair Proteins In Vivo
  • 10. Conclusions
  • Acknowledgments
  • References
  • Section IV: Hydrogen-Deuterium Exchange Mass Spectrometry
  • Chapter Thirteen: Hydrogen Exchange Mass Spectrometry
  • 1. Introduction
  • 2. HX Mechanism
  • 2.1. Chemistry
  • 2.2. HX Structural Physics
  • 3. Experimental Considerations-Exchange Labeling
  • 3.1. Continuous Labeling (Native State)
  • 3.1.1. Binding Sites and Epitope Mapping
  • 3.2. Pulsed Labeling
  • 4. Experimental Considerations-MS Measurement
  • 4.1. Whole Protein
  • 4.2. Peptide Resolved
  • 4.2.1. Proteolysis
  • 4.2.2. Chromatography
  • 5. Mass Spectrometer Instrument Considerations
  • 6. Data Analysis
  • 6.1. Whole Protein
  • 6.2. Peptide Analysis
  • 6.2.1. Identification of Labeled Peptides
  • 6.2.2. Back Exchange Correction
  • 6.3. Toward Residue Resolution
  • 6.4. Analysis of Pulsed Exchange Data
  • 7. Conclusion
  • References
  • Chapter Fourteen: Mapping Protein-Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Sp ...
  • 1. Introduction
  • 2. H/D Exchange Theory
  • 2.1. H/D Exchange
  • 2.2. Acid and Base Catalysis
  • 2.3. Temperature
  • 2.4. Protein Structure
  • 2.5. Effects of Ligand-Binding Interactions upon H/D Exchange Rates
  • 3. The HDX-MS Experiment
  • 3.1. Synopsis
  • 3.2. Protein Preparation
  • 3.3. Exchange Reaction
  • 3.4. Automated Versus Manual HDX-MS
  • 3.5. Reaction Quenching
  • 3.6. Enzymatic Digestion
  • 3.7. Chromatography
  • 3.8. Mass Spectrometry
  • 3.9. Generating a Proteomic Map
  • 3.10. Data Analysis
  • 3.11. Uncertainty Evaluations: Which Deuterium-Uptake Differences Are Meaningful?
  • 3.12. Data Display
  • 4. Interpreting HDX-MS Data to Determine Protein-Ligand Interaction Maps
  • 4.1. Example: Protein-Ligand Interactions Involving Continuous Amide Contacts
  • 4.2. Example: Mapping a Discontinuous Protein-Protein Interaction
  • Disclaimer
  • References
  • Chapter Fifteen: Isotope Labeling of Biomolecules: Structural Analysis of Viruses by HDX-MS
  • 1. Introduction
  • 2. Methods
  • 2.1. Initial Considerations for HDX-MS and Sample Requirements
  • 2.2. Deuterium Exchange Reaction
  • 2.2.1. Quenching the Exchange
  • 2.2.2. Digestion Optimization and Peptide Identification
  • 2.2.3. Additional HDX Controls
  • 2.2.4. Liquid Chromatography-Mass Spectrometry
  • 2.2.5. Peptide Identification
  • 2.2.6. HDX Data Analysis
  • 3. Example HDX-MS Experiment with Bovine Ribonuclease A
  • 3.1. Deuteration and Quenching
  • Acknowledgments
  • References
  • Author Index
  • Subject Index
  • Color Plate
  • Back Cover

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