Non-Natural Amino Acids

1. Auflage 2009
ISBN: 978-0-08-092163-1
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 334 Seiten

By combining the tools of organic chemistry with those of physical biochemistry and cell biology, Non-Natural Amino Acids aims to provide fundamental insights into how proteins work within the context of complex biological systems of biomedical interest.
The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. With more than 400 volumes published, each Methods in Enzymology volume presents material that is relevant in today's labs -- truly an essential publication for researchers in all fields of life sciences.
Demonstrates how the tools and principles of chemistry combined with the molecules and processes of living cells can be combined to create molecules with new properties and functions found neither in nature nor in the test tube
Presents new insights into the molecular mechanisms of complex biological and chemical systems that can be gained by studying the structure and function of non-natural molecules
Provides a 'one-stop shop' for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods

Autoren/Hrsg.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;10
6;Preface;13
7;Methods in Enzymology;17
8;Chapter 1: Protein Phosphorylation by Semisynthesis: From Paper to Practice;45
8.1;1. Overview of Protein Phosphorylation;46
8.2;2. Investigating Protein Phosphorylation with Phosphomimetics;47
8.2.1;2.1. Thiophosphate substitution as a phosphomimetic;48
8.2.2;2.2. Using amino acid substitution as a phosphomimetic;48
8.2.3;2.3. Incorporation of alternative genetically encoded phosphomimetics;48
8.3;3. Phosphonate Analogues and Protein Semisynthesis;49
8.4;4. Methods;50
8.4.1;4.1. Choosing a protein for semisynthesis;52
8.4.2;4.2. Choosing the peptide ligation site;52
8.4.3;4.3. Design of recombinant constructs for semisynthesis;53
8.4.4;4.4. Peptide synthesis;54
8.4.5;4.5. C-terminal semisynthesis (EPL) on a soluble protein;55
8.4.6;4.6. C-terminal EPL on an insoluble protein;57
8.4.7;4.7. N-terminal semisynthesis;58
8.4.8;4.8. Purification of the semisynthetic protein;58
8.5;5. Practical Uses of Semisynthetic Phosphoproteins;58
8.5.1;5.1. Kinetic analysis of phosphonylated enzymes;58
8.5.2;5.2. Microinjection of phosphonylated enzymes;60
8.5.3;5.3. Pull-down assays using phosphonylated enzymes as bait;63
8.6;6. Future of Protein Semisynthesis in Signaling;63
8.7;Acknowledgments;65
8.8;References;65
9;Chapter 2: Protein Engineering with the Traceless Staudinger Ligation;69
9.1;1. Introduction;70
9.2;2. Traceless Staudinger Ligation;70
9.3;3. Choice of Coupling Reagent;72
9.3.1;3.1. Experimental procedure: Synthesis of phosphinothiol I;75
9.4;4. Preparation of the Azido Fragment;77
9.4.1;4.1. Experimental procedure: Strategy N1;78
9.5;5. Preparation of the Phosphinothioester Fragment;79
9.5.1;5.1. Experimental procedure: Strategy C1;82
9.5.2;5.2. Experimental procedure: Strategy C5;83
9.6;6. Protein Assembly by Orthogonal Chemical Ligations;84
9.6.1;6.1. Experimental procedure: Traceless Staudinger ligation on a solid phase;84
9.7;7. Prospectus;85
9.7.1;Experimental procedure: General;86
9.8;Acknowledgments;87
9.9;References;87
10;Chapter 3: Replacement of Y730 and Y731 in the alpha2 Subunit of Escherichia coli Ribonucleotide Reductase with 3-Aminotyrosine using and Evolved Suppressor tRNA/tRNA- Synthetase Pair;89
10.1;1. Introduction;91
10.2;2. Site-Specific Insertion of Unnatural Amino Acids using the Suppressor tRNA/RS Method;93
10.3;3. NH2Y, a Y Analogue for Investigating Enzymatic PCET Reactions;95
10.3.1;3.1. Overview: Choice of NH2Y;95
10.3.2;3.2. Protocol for assessing uptake and toxicity of NH2Y in E. coli;95
10.3.3;3.3. Results;96
10.4;4. Directed Evolution of NH2Y-RS in E. coli;97
10.5;5. Examination of the Fidelity and Specificity of NH2Y Incorporation in a Protein Expressed in E. coli;99
10.5.1;5.1. Overview: The Z-domain as a model;99
10.5.2;5.2. Protocol for incorporation of NH2Y into the Z-domain;99
10.5.3;5.3. Results;100
10.6;6. Generation of Y730NH2Y-alpha2 and Y731NH2Y-alpha2;101
10.6.1;6.1. Overview;101
10.6.2;6.2. Unsuccessful attempts to incorporate NH2Y into alpha2;101
10.6.3;6.3. Successful incorporation of NH2Y into alpha2;103
10.6.4;6.4. Protocol for successful expression of NH2Y-alpha2s;103
10.6.5;6.5. Results;104
10.6.6;6.6. Protocol for purification of NH2Y-alpha2s;104
10.6.7;6.7. Assessment of the fidelity and specificity of NH2Y insertion into alpha2;105
10.6.8;6.8. Protocol for measurement of catalytic activities of NH2Y-alpha2s;107
10.6.9;6.9. Protocol for use of the mechanism-based RNR inhibitor, N3ADP;107
10.6.10;6.10. Results of activity and N3ADP assays;108
10.7;7. Characterization of NH2Y-alpha2s;109
10.7.1;Overview;109
10.7.2;Protocol for determining the EPR and UV-vis properties of NH2Y.-alpha2s;110
10.7.3;Results;110
10.7.4;Protocol for determining the kinetics of NH2Y.-alpha2 formation by SF UV-vis spectroscopy;112
10.7.5;Protocol for determining the kinetics of NH2Y.-alpha2 formation by RFQ EPR spectroscopy;112
10.7.6;Results of SF UV-vis and RFQ EPR spectroscopic studies;113
10.8;8. Summary;115
10.9;Acknowledgments;115
10.10;References;115
11;Chapter 4: Semisynthesis of Proteins Using Split Inteins;121
11.1;1. Introduction;122
11.2;2. Protein Splicing in cis and in trans is Performed by Inteins;125
11.3;3. Design of Split Inteins and Considerations on the Intein Insertion Site;126
11.4;4. Materials and Methods;131
11.4.1;4.1. Solid-phase peptide synthesis of ExN-IntN peptides;131
11.4.2;4.2. Construction of IntC-POI plasmids;132
11.4.3;4.3. Expression and purification of IntC-POI fusion proteins;132
11.4.4;4.4. Protein trans-splicing assays;133
11.4.5;4.5. Interaction studies of the IntN/IntC association;134
11.4.6;4.6. Purification and functional analysis of semisynthetic splice products;135
11.4.7;4.7. Protein splicing in complex mixtures;135
11.5;5. Summary and Conclusion;136
11.6;Acknowledgments;137
11.7;References;137
12;Chapter 5: Expressed Protein Ligation for Metalloprotein Design and Engineering;141
12.1;1. Introduction;160
12.2;2. Methods of Selenocysteine Incorporation;163
12.3;3. Incorporation of Selenocysteine into the Type 1 Copper Site of Azurin;164
12.3.1;3.1. Synthesis of Fmoc-Sec(PMB)-OH;165
12.3.2;3.2. Solid-phase peptide synthesis (SPPS) of C-terminal 17-mer peptide;166
12.3.3;3.3. Deprotection of selenocysteine-containing 17-mer peptide;167
12.3.4;3.4. General procedure for EPL of Azurin(1-111)- Intein-CBD and the C-terminal 17-mer peptide;169
12.3.5;3.5. Characterization of selenocysteine azurin;170
12.4;4. Tuning the Type 1 Copper Reduction Potential Using Isostructural Methionine Analogues;171
12.4.1;4.1. Use of EPL for incorporation of methionine analogues;171
12.4.2;4.2. General procedure for the SPPS of methionine analogue 17-mer peptides;171
12.4.3;4.3. General procedure for the peptide cleavage from the resin;172
12.4.4;4.4. General procedure for EPL of azurin(1-111)-intein-CBD and the C-terminal peptides containing methionie analogues;172
12.4.5;4.5. Characterization of methionine analogues of azurin;173
12.5;5. Conclusion;173
12.6;Acknowledgments;174
12.7;References;175
13;Chapter 6: Using Expressed Protein Ligation to Probe the Substrate Specificity of Lantibiotic Synthetases;142
13.1;1. Introduction;143
13.2;2. Use of Expressed Protein Ligation to Prepare Substrate Analogues;145
13.2.1;2.1. Overview;145
13.2.2;2.2. General procedure for solid-phase peptide synthesis;148
13.2.3;2.3. General procedure for purification of the peptide thioester His-LctA (1-37)-MES;149
13.2.4;2.4. General procedure for ligation of LctA(1-37)MES with short peptides;150
13.2.5;2.5. Investigation of the dehydration reaction;151
13.2.6;2.6. General procedure for LctM dehydration assays with truncated LctA analogues;152
13.2.7;2.7. Procedure for LctM-catalyzed dehydration of LctA(1-43) with difluoromethylthreonine (8) at position 42;153
13.2.8;2.8. Investigation of the cyclization reaction;153
13.2.9;2.9. General procedure for LctM and LctM-C781A assays with truncated LctA analogues and analysis of the assay products with CNBr;155
13.3;3. Conclusion;157
13.4;Acknowledgments;158
13.5;References;158
14;Chapter 7: Semisynthesis of K+ Channels;197
14.1;1. Introduction;198
14.2;2. Experimental Protocols;201
14.2.1;2.1. Synthetic design;201
14.2.2;2.2. Synthesis of the KcsA 70-123 (pF-Phe103) C-peptide;202
14.2.3;2.3. Generation of the N-peptide thioester;204
14.2.4;2.4. The ligation reaction;207
14.2.5;2.5. Folding of the semisynthetic KcsA channel;207
14.2.6;2.6. Functional characterization of the semisynthetic KcsA;208
14.3;3. Application of Semisynthesis in Investigating the Selectivity Filter of the K+ Channels;209
14.3.1;3.1. D-Ala substitution in the selectivity filter (Valiyaveetil et al., 2004, 2006);209
14.3.2;3.2. Ester substitution in the selectivity filter (Valiyaveetil et al., 2006);210
14.4;4. Summary;210
14.5;Acknowledgments;211
14.6;References;211
15;Chapter 8: Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance;213
15.1;1. Introduction;214
15.2;2. Segmental Isotopic Labeling using Expressed Protein Ligation;215
15.2.1;2.1. Overview;215
15.2.2;2.2. Ligation site;217
15.2.3;2.3. Synthesis of a segment with C-terminal alpha- thioester;220
15.2.4;2.4. Synthesis of a segment with N-terminal cysteine;220
15.2.5;2.5. Ligation protocol;221
15.3;3. Segmental Labeling using Protein Trans-Splicing;221
15.4;4. Multiple Segment Assembly;222
15.5;5. Segmental Labeling of C-Terminal SRC Kinase(Csk);222
15.5.1;5.1. Overview;222
15.5.2;5.2. Cloning Csk SH32 and kinase gene to expression vector;225
15.5.3;5.3. Expression and purification Csk SH32 with C-terminal Mxe GyrA intein (intein2);226
15.5.4;5.4. Expression and purification of Csk kinase with N-terminal Ssp DnaB intein (intein1);227
15.5.5;5.5. Ligation of SH32 domain with testing peptide;229
15.5.6;5.6. Ligation of SH32 with kinase domain;230
15.5.7;5.7. Purification of ligation product;230
15.5.8;5.8. NMR spectroscopy;232
15.6;Acknowledgments;233
15.7;References;233
16;Chapter 9: Semisynthesis of Membrane-Attached Prion Proteins;239
16.1;1. Introduction;240
16.2;2. Chemical Synthesis of Membrane Anchors;241
16.3;3. Semisynthesis of rPrPPalm by Expressed Protein Ligation;242
16.3.1;3.1. Cloning procedure;243
16.3.2;3.2. Bacterial expression and protein purification;244
16.3.3;3.3. Intein cleavage and purification;244
16.3.4;3.4. Native chemical ligation reactions;245
16.3.5;3.5. Folding of rPrPPalm and rPrPGPI;246
16.3.6;3.6. Comments;247
16.4;4. Semisynthesis of rPrPPalm by Protein Trans-Splicing;247
16.4.1;4.1. Cloning procedure;247
16.4.2;4.2. Bacterial expression and protein purification;247
16.4.3;4.3. Refolding of rPrP-DnaEN-His;248
16.4.4;4.4. Synthesis of DnaEC-membrane anchor peptides;248
16.4.5;4.5. Trans-splicing with rPrP-DnaEN-His;249
16.4.6;4.6. Folding of rPrPPalm;249
16.4.7;4.7. Comments;250
16.5;5. Liposome Attachment and Aggregation of rPrPPalm and rPrPGPI;250
16.5.1;5.1. Vesicle attachment of rPrPPalm and rPrPGPI;251
16.5.2;5.2. Aggregation assays;251
16.6;6. Conclusion;252
16.7;References;252
17;Chapter 10: Use of Intein-Mediated Protein Ligation Strategies for the Fabrication of Functional Protein Arrays;257
17.1;1. Introduction;258
17.2;2. Intein-Mediated Protein Ligation Strategies;259
17.2.1;2.1. N-terminal intein fusions for protein immobilization;261
17.2.2;2.2. C-terminal intein fusions for protein biotinylation and immobilization;264
17.3;3. In Vitro, In Vivo and Cell Free Strategies for Protein Biotinylation at the C-Terminal;265
17.4;4. Methods;269
17.4.1;4.1. Protocols for biotinylation of proteins at the C-terminus;269
17.4.1.1;4.1.1. In vitro protein biotinylation;269
17.4.1.1.1;4.1.1.1. Practical considerations;271
17.4.1.2;4.1.2. In vivo protein biotinylation;277
17.4.1.3;4.1.3. Cell-free protein expression and biotinylation;277
17.4.2;4.2. Protocol for generation of N-terminal cysteine-containing proteins;278
17.4.3;4.3. Preparation of slides;279
17.4.3.1;4.3.1. Thioester slides;280
17.4.3.2;4.3.2. Avidin slides;280
17.4.4;4.4. Spotting of slides and detection of proteins;280
17.5;5. Concluding Remarks;281
17.6;References;282
18;Chapter 11: Semisynthesis of Ubiquitylated Proteins;287
18.1;1. Introduction;288
18.2;2. Semisynthesis of Ubiquitylated Histone H2B;290
18.2.1;2.1. Overall synthetic design;290
18.3;3. Methods;292
18.3.1;3.1. General methods;292
18.4;4. Synthesis of Photocleavable Ligation Auxiliary;293
18.4.1;4.1. 4-(2-Methoxy-5-nitro-4-vinyl-phenoxy)-butyric acid methyl ester (2);293
18.4.2;4.2. 4-[4-(1-tert-Butoxycarbonylamino-2-hydroxy-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid methyl easter (3);294
18.4.3;4.3. 4-[4-(2-Acetylsulfanyl-1-tert-butoxycarbonylamino-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid mehyl ester (4);295
18.4.4;4.4. 4-[4-(1-tert-Butoxycarbonylamino-2-tert-butyldisulfanyl-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid (5);295
18.4.5;4.5. 4-[4-(1-Amino-2-tert-butyldisulfanyl-ethyl)-2-methoxy-5-nitro-phenoxy]-N-methyl-butyramide (6);296
18.5;5. Peptide Synthesis;296
18.5.1;5.1. Chemical synthesis of peptide 7;297
18.6;6. Generation of Recombinant Protein alpha- Thioesters;297
18.6.1;6.1. Preparation of ubiquitin(1-75)- alpha- MES (8);298
18.6.2;6.2. Preparation of H2B(1-116)-alpha- MES (9);298
18.7;7. Expressed Protein Ligation;299
18.7.1;7.1. Synthesis of ubiquitylated peptide 10;300
18.7.2;7.2. Photolytic deprotection of 10 to give branched protein 11;300
18.7.3;7.3. Synthesis of ubiquitylated H2B mutant 12;301
18.7.4;7.4. Desulfurization of 12 to give uH2B 13;301
18.8;8. Generation of Ubiquitylated Mononucleosomes;301
18.8.1;8.1. Recombinant histone preparation;302
18.8.2;8.2. Preparation of DNA for nucleosome formation;303
18.8.3;8.3. Ubiquitylated nucleosome formation;303
18.9;9. Conclusions;304
18.10;Acknowledgments;304
18.11;References;304
19;Author Index;307
20;Subject Index;321
21;Color Plate;327