1;Preface;5 2;Contents;7 3;Contributors;9 4;Understanding Enzyme Mechanism through Protein Chimeragenesis;12 4.1;1 Introduction;13 4.2;2 Chimeragenesis Methods;15 4.3;3 Case Studies;21 4.4;4 Conclusion;33 4.5;References;35 5;Chemical Protein Engineering: Synthetic and Semisynthetic Peptides and Proteins;39 5.1;1 Introduction;40 5.2;2 Synthesis of Peptides and Proteins;40 5.3;3 Chemical Modification of Proteins;54 5.4;4 Enzyme-Mediated Peptide Bond Formation;65 5.5;References;68 6;Native Chemical Ligation: SemiSynthesis of Post- translationally Modified Proteins and Biological Probes;75 6.1;1 Introduction;76 6.2;2 Engineering Design Considerations for NCL;77 6.3;3 Thioester Generation;80 6.4;4 N-Terminal Cysteine Fragments;84 6.5;5 Extensions of NCL;85 6.6;6 Applications;87 6.7;7 Conclusion;102 6.8;References;102 7;Chemical Methods for Mimicking Post- Translational Modifications;107 7.1;1 Introduction;108 7.2;2 Glycosylation;112 7.3;3 PEGylation as a Mimic of Glycosylation;122 7.4;4 Lipidation;125 7.5;5 Phosphorylation;127 7.6;6 Sulfation;127 7.7;7 Conclusion;128 7.8;References;129 8;Noncanonical Amino Acids in Protein Science and Engineering;136 8.1;1 Incorporation of Noncanonical Amino Acids into Engineered Proteins;137 8.2;2 Translational Fidelity: Aminoacyl-tRNA Synthetases;140 8.3;3 Reactive Amino Acids;140 8.4;4 Fluorescent Amino Acids;146 8.5;5 Photosensitive Amino Acids;149 8.6;6 Fluorinated Amino Acids;153 8.7;7 Conclusion;155 8.8;References;155 9;Fidelity Mechanisms of the Aminoacyl- tRNA Synthetases;163 9.1;1 Abstract;163 9.2;2 Aminoacylation and Specificity;164 9.3;3 CP1 Domain Based Editing - IleRS, ValRS, and LeuRS;182 9.4;4 Class II Synthetase Editing Mechanisms;188 9.5;5 Single Site Editing and Cyclization;195 9.6;6 Loss of Editing Function in AARSs;196 9.7;7 Role of AARS Editing In Vivo;197 9.8;8 Orthogonal AARS and tRNAs;198 9.9;9 Adapting AARS Editing Sites for Protein Engineering Applications;200 9.10;References;201 10;Specialized Components of the Translational Machinery for Unnatural Amino Acid Mutagenesis: tRNAs, Aminoacyl- tRNA Synthetases, and Ribosomes;212 10.1;1 Introduction;212 10.2;2 Basic Principles of Unnatural Amino Acid Mutagenesis;213 10.3;3 Engineering the Perfect Host for Unnatural Amino Acid Mutagenesis: Optimization and Fine- Tuning of Unnatural Amino Acid Mutagenesis by Manipulating Individual Components of the Translational Machinery;223 10.4;4 Conclusion;231 10.5;References;232 11;In Vivo Studies of Receptors and Ion Channels with Unnatural Amino Acids;237 11.1;Contents;237 11.2;1 Introduction;238 11.3;2 In Vivo Nonsense Suppression Method for Unnatural Amino Acid Incorporation;239 11.4;3 "Highly Unnatural" Amino Acids;244 11.5;4 Structure-Function Studies;251 11.6;5 Conclusion;255 11.7;References;256 12;Synthesis of Modified Proteins Using Misacylated tRNAs;261 12.1;Contents;261 12.2;1 Introduction;262 12.3;2 Amino Acid Protecting Groups for Misacylated tRNAs;262 12.4;3 Alternative Codons for Incorporation of Unnatural Amino Acids;266 12.5;4 Incorporation of Unnatural Amino Acids Affording Proteins Having Modified Backbones;267 12.6;5 Bisaminoacylated tRNAs as Participants in Protein Synthesis;271 12.7;References;273 13;Cell-Free Synthesis of Proteins with Unnatural Amino Acids. The PURE System and Expansion of the Genetic Code;277 13.1;Contents;277 13.2;1 Introduction;278 13.3;2 Conventional Cell-Free Translation;278 13.4;3 PURE System;281 13.5;4 Expansion of the Genetic Code;285 13.6;5 Conclusion;292 13.7;References;293 14;Engineering Nucleobases and Polymerases for an Expanded Genetic Alphabet;297 14.1;1 Introduction;298 14.2;2 Unnatural DNA Base Pairs;302 14.3;3 Recognition of Unnatural DNA Base Pairs by DNA Polymerases: Alternatives to Kf;311 14.4;4 Conclusion;316 14.5;References;316 15;Understanding Membrane Proteins. How to Design Inhibitors of Transmembrane Protein- Protein Interactions;320 15.1;1 Introduction: The Significance of and Impediments to Designing Transmembrane Prote