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.