Protein Engineering (eBook)

Preface 5
Contents 7
Contributors 9
Understanding Enzyme Mechanism through Protein Chimeragenesis 12
1 Introduction 13
2 Chimeragenesis Methods 15
3 Case Studies 21
4 Conclusion 33
References 35
Chemical Protein Engineering: Synthetic and Semisynthetic Peptides and Proteins 39
1 Introduction 40
2 Synthesis of Peptides and Proteins 40
3 Chemical Modification of Proteins 54
4 Enzyme-Mediated Peptide Bond Formation 65
References 68
Native Chemical Ligation: SemiSynthesis of Post- translationally Modified Proteins and Biological Probes 75
1 Introduction 76
2 Engineering Design Considerations for NCL 77
3 Thioester Generation 80
4 N-Terminal Cysteine Fragments 84
5 Extensions of NCL 85
6 Applications 87
7 Conclusion 102
References 102
Chemical Methods for Mimicking Post- Translational Modifications 107
1 Introduction 108
2 Glycosylation 112
3 PEGylation as a Mimic of Glycosylation 122
4 Lipidation 125
5 Phosphorylation 127
6 Sulfation 127
7 Conclusion 128
References 129
Noncanonical Amino Acids in Protein Science and Engineering 136
1 Incorporation of Noncanonical Amino Acids into Engineered Proteins 137
2 Translational Fidelity: Aminoacyl-tRNA Synthetases 140
3 Reactive Amino Acids 140
4 Fluorescent Amino Acids 146
5 Photosensitive Amino Acids 149
6 Fluorinated Amino Acids 153
7 Conclusion 155
References 155
Fidelity Mechanisms of the Aminoacyl- tRNA Synthetases 163
1 Abstract 163
2 Aminoacylation and Specificity 164
3 CP1 Domain Based Editing – IleRS, ValRS, and LeuRS 182
4 Class II Synthetase Editing Mechanisms 188
5 Single Site Editing and Cyclization 195
6 Loss of Editing Function in AARSs 196
7 Role of AARS Editing In Vivo 197
8 Orthogonal AARS and tRNAs 198
9 Adapting AARS Editing Sites for Protein Engineering Applications 200
References 201
Specialized Components of the Translational Machinery for Unnatural Amino Acid Mutagenesis: tRNAs, Aminoacyl- tRNA Synthetases, and Ribosomes 212
1 Introduction 212
2 Basic Principles of Unnatural Amino Acid Mutagenesis 213
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
4 Conclusion 231
References 232
In Vivo Studies of Receptors and Ion Channels with Unnatural Amino Acids 237
Contents 237
1 Introduction 238
2 In Vivo Nonsense Suppression Method for Unnatural Amino Acid Incorporation 239
3 “Highly Unnatural” Amino Acids 244
4 Structure–Function Studies 251
5 Conclusion 255
References 256
Synthesis of Modified Proteins Using Misacylated tRNAs 261
Contents 261
1 Introduction 262
2 Amino Acid Protecting Groups for Misacylated tRNAs 262
3 Alternative Codons for Incorporation of Unnatural Amino Acids 266
4 Incorporation of Unnatural Amino Acids Affording Proteins Having Modified Backbones 267
5 Bisaminoacylated tRNAs as Participants in Protein Synthesis 271
References 273
Cell-Free Synthesis of Proteins with Unnatural Amino Acids. The PURE System and Expansion of the Genetic Code 277
Contents 277
1 Introduction 278
2 Conventional Cell-Free Translation 278
3 PURE System 281
4 Expansion of the Genetic Code 285
5 Conclusion 292
References 293
Engineering Nucleobases and Polymerases for an Expanded Genetic Alphabet 297
1 Introduction 298
2 Unnatural DNA Base Pairs 302
3 Recognition of Unnatural DNA Base Pairs by DNA Polymerases: Alternatives to Kf 311
4 Conclusion 316
References 316
Understanding Membrane Proteins. How to Design Inhibitors of Transmembrane Protein– Protein Interactions 320
1 Introduction: The Significance of and Impediments to Designing Transmembrane Protein Inhibitors 321
2 Considerations for Peptide Design, Part I: How Amino Acids Modulate Transmembrane Structure 323
3 Considerations for Peptide Design, Part II: Forces Responsible for Oligomerization 329
4 Methods for Design: Computational Potential Energy Functions 332
5 Putting It All Together: Membrane Structure Prediction and Membrane Protein Design 335
6 Conclusion 337
References 337
Index 343