Unnatural Amino Acids Methods and Protocols

About this book

Even though they are present in nature, non-proteinogenic amino acids are usually defined as unnatural or non-natural. Beside their structural diversity, interest in these compounds is due to their occurrence in nature, their biological properties, the analytical aspects, their use as probes, and their incorporation into peptides and proteins, among other reasons. Divided into five convenient sections, "Unnatural Amino Acids: Methods and Protocols" deals with enzymatic methods used to produce non-natural amino acids, aspects concerning the presence of unnatural amino acids in peptides with antimicrobial properties, genetic incorporation of unnatural amino acids into proteins (yeast and mammalian cells), and detection and quantification of D-amino acids and related enzymes.

Written in the highly successful "Methods in Molecular Biology" series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, "Unnatural Amino Acids: Methods and Protocols" serves as an ideal guide for scientists and contributes to directing the attention of researchers to the many fields of growing scientific interest in non-natural amino acids.

Contents

Part I. Synthesis of Unnatural Amino Acids
1. Preparation of Unnatural Amino Acids with Ammonia-lyases and 2,3-aminomutases
2. Multistep Enzyme Catalyzed Reactions for Unnatural Amino Acids
3. Enzymatic Production of Enantiopure Amino Acids from Mono-substituted Hydantoin Substrates
4. Preparation of Glutamate Analogues by Enzymatic Transamination
5. Carbon-Carbon Bond Forming Enzymes for the Synthesis of Non-natural Amino Acids
6. Engineering Cyclic Amidases for Non-natural Amino Acid Synthesis


Part II. Application of Unnatural Amino Acids
7. NMR Analysis of Unnatural Amino Acids in Natural Antibiotics
8. Site-specific Incorporation of Unnatural Amino Acids as Probes for Protein Conformational Changes
9. Application of Unnatural Amino Acids to the De novo Design of Selective Antibiotic Peptides
10. Use of Unnatural Amino Acids to Probe Structure-activity Relationships and Mode-of-action of Antimicrobial Peptides

Part III. Use of Unnatural Amino Acids in Protein Synthesis
11. Experimental Methods for Scanning Unnatural Amino Acid Mutagenesis
12. Genetic Incorporation of Unnatural Amino Acids into Proteins in Yeast
13. Site-specific Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells
14. Incorporation of Unnatural Non-a-amino Acids into the N-terminus of Proteins in a Cell-free Translation System
15. Site Specific Modification of Proteins by the Staudinger-phosphite Reaction

Part IV. D-amino Acids: Analysis and Applications
16. HPLC Methods for Determination of D-aspartate and N-methyl-D-aspartate
17. Estimation of Chronological Age from the Racemization Rate of L- and D-aspartic Acid: How to Completely Separate Enantiomers from Dentin
18. Enzymatic Detection of D-amino Acids
19. An Enzymatic-HPLC Assay to Monitor Endogenous D-serine Release from Neuronal Cultures
20. Electrophysiological Analysis of the Modulation of NMDA-receptors Function by d-serine and Glycine in the Central Nervous System
21. Biosensors for D-amino Acid Detection
22. Analysis of D-#-aspartyl Isomers at Specific Sites in Proteins
23. Nutritional Value of D-amino Acids, D-peptides and Amino Acid Derivatives in Mice

Part V. Enzymes Active on D-amino Acids
24. Preparation and Assay of Recombinant Serine Racemase
25. Assay of Amino Acid Racemases
26. Assays of D-amino Acid Oxidases
27. Enzymes Acting on D-amino Acid Containing Peptides

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