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• Cover Image
• Table of Contents
• Preface
• Chapter 1 Protein Phosphorylation by Semisynthesis
• 1. Overview of Protein Phosphorylation
• 2. Investigating Protein Phosphorylation with Phosphomimetics
• 3. Phosphonate Analogues and Protein Semisynthesis
• 4. Methods
• 5. Practical Uses of Semisynthetic Phosphoproteins
• 6. Future of Protein Semisynthesis in Signaling
• Acknowledgments
• Chapter 2 Protein Engineering with the Traceless Staudinger Ligation
• 1. Introduction
• 2. Traceless Staudinger Ligation
• 3. Choice of Coupling Reagent
• 4. Preparation of the Azido Fragment
• 5. Preparation of the Phosphinothioester Fragment
• 6. Protein Assembly by Orthogonal Chemical Ligations
• 7. Prospectus
• Acknowledgments
• Chapter 3 Replacement of Y730 and Y731 in the α2 Subunit of Escherichia coli Ribonucleotide Reductase with 3‐Aminotyrosine using an Evolved Suppressor tRNA/tRNA‐Synthetase Pair
• 1. Introduction
• 2. Site‐Specific Insertion of Unnatural Amino Acids using the Suppressor tRNA/RS Method
• 3. NH2Y, a Y Analogue for Investigating Enzymatic PCET Reactions
• 4. Directed Evolution of NH2Y‐RS in E. coli
• 5. Examination of the Fidelity and Specificity of NH2Y Incorporation in a Protein Expressed in E. coli
• 6. Generation of Y730NH2Y‐α2 and Y731NH2Y‐α2
• 7. Characterization of NH2Y‐α2s
• 8. Summary
• Acknowledgements
• Chapter 4 Semisynthesis of Proteins Using Split Inteins
• 1. Introduction
• 2. Protein Splicing in cis and in trans is Performed by Inteins
• 3. Design of Split Inteins and Considerations on the Intein Insertion Site
• 4. Materials and Methods
• 5. Summary and Conclusion
• Acknowledgments
• Chapter 5 Expressed Protein Ligation for Metalloprotein Design and Engineering
• 1. Introduction
• 2. Methods of Selenocysteine Incorporation
• 3. Incorporation of Selenocysteine into the Type 1 Copper Site of Azurin
• 4. Tuning the Type 1 Copper Reduction Potential Using Isostructural Methionine Analogues
• 5. Conclusion
• Acknowledgments
• Chapter 6 Using Expressed Protein Ligation to Probe the Substrate Specificity of Lantibiotic Synthetases
• 1. Introduction
• 2. Use of Expressed Protein Ligation to Prepare Substrate Analogues
• 3. Conclusion
• Acknowledgments
• Chapter 7 Semisynthesis of K+ Channels
• 1. Introduction
• 2. Experimental Protocols
• 3. Application of Semisynthesis in Investigating the Selectivity Filter of the K+ Channels
• 4. Summary
• Acknowledgment
• Chapter 8 Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance
• 1. Introduction
• 2. Segmental Isotopic Labeling using Expressed Protein Ligation
• 3. Segmental Labeling using Protein Trans‐Splicing
• 4. Multiple Segment Assembly
• 5. Segmental Labeling of C‐Terminal SRC Kinase(Csk)
• Acknowledgments
• Chapter 9 Semisynthesis of Membrane‐Attached Prion Proteins
• 1. Introduction
• 2. Chemical Synthesis of Membrane Anchors
• 3. Semisynthesis of rPrPPalm by Expressed Protein Ligation
• 4. Semisynthesis of rPrPPalm by Protein Trans‐Splicing
• 5. Liposome Attachment and Aggregation of rPrPPalm and rPrPGPI
• 6. Conclusion
• Chapter 10 Use of Intein‐Mediated Protein Ligation Strategies for the Fabrication of Functional Protein Arrays
• 1. Introduction
• 2. Intein‐Mediated Protein Ligation Strategies
• 3. In Vitro, In Vivo and Cell Free Strategies for Protein Biotinylation at the C‐Terminal
• 4. Methods
• 5. Concluding Remarks
• Chapter 11 Semisynthesis of Ubiquitylated Proteins
• 1. Introduction
• 2. Semisynthesis of Ubiquitylated Histone H2B
• 3. Methods
• 4. Synthesis of Photocleavable Ligation Auxiliary
• 5. Peptide Synthesis
• 6. Generation of Recombinant Protein α‐Thioesters
• 7. Expressed Protein Ligation
• 8. Generation of Ubiquitylated Mononucleosomes
• 9. Conclusions
• Acknowledgments