Non-Natural Amino Acids (eBook)

Front Cover 1
Methods in Enzymology 4
Copyright Page 5
Contents 6
Contributors 10
Preface 13
Methods in Enzymology 17
Chapter 1: Protein Phosphorylation by Semisynthesis: From Paper to Practice 45
1. Overview of Protein Phosphorylation 46
2. Investigating Protein Phosphorylation with Phosphomimetics 47
2.1. Thiophosphate substitution as a phosphomimetic 48
2.2. Using amino acid substitution as a phosphomimetic 48
2.3. Incorporation of alternative genetically encoded phosphomimetics 48
3. Phosphonate Analogues and Protein Semisynthesis 49
4. Methods 50
4.1. Choosing a protein for semisynthesis 52
4.2. Choosing the peptide ligation site 52
4.3. Design of recombinant constructs for semisynthesis 53
4.4. Peptide synthesis 54
4.5. C-terminal semisynthesis (EPL) on a soluble protein 55
4.6. C-terminal EPL on an insoluble protein 57
4.7. N-terminal semisynthesis 58
4.8. Purification of the semisynthetic protein 58
5. Practical Uses of Semisynthetic Phosphoproteins 58
5.1. Kinetic analysis of phosphonylated enzymes 58
5.2. Microinjection of phosphonylated enzymes 60
5.3. Pull-down assays using phosphonylated enzymes as bait 63
6. Future of Protein Semisynthesis in Signaling 63
Acknowledgments 65
References 65
Chapter 2: Protein Engineering with the Traceless Staudinger Ligation 69
1. Introduction 70
2. Traceless Staudinger Ligation 70
3. Choice of Coupling Reagent 72
3.1. Experimental procedure: Synthesis of phosphinothiol I 75
4. Preparation of the Azido Fragment 77
4.1. Experimental procedure: Strategy N1 78
5. Preparation of the Phosphinothioester Fragment 79
5.1. Experimental procedure: Strategy C1 82
5.2. Experimental procedure: Strategy C5 83
6. Protein Assembly by Orthogonal Chemical Ligations 84
6.1. Experimental procedure: Traceless Staudinger ligation on a solid phase 84
7. Prospectus 85
Experimental procedure: General 86
Acknowledgments 87
References 87
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
1. Introduction 91
2. Site-Specific Insertion of Unnatural Amino Acids using the Suppressor tRNA/RS Method 93
3. NH2Y, a Y Analogue for Investigating Enzymatic PCET Reactions 95
3.1. Overview: Choice of NH2Y 95
3.2. Protocol for assessing uptake and toxicity of NH2Y in E. coli 95
3.3. Results 96
4. Directed Evolution of NH2Y-RS in E. coli 97
5. Examination of the Fidelity and Specificity of NH2Y Incorporation in a Protein Expressed in E. coli 99
5.1. Overview: The Z-domain as a model 99
5.2. Protocol for incorporation of NH2Y into the Z-domain 99
5.3. Results 100
6. Generation of Y730NH2Y-alpha2 and Y731NH2Y-alpha2 101
6.1. Overview 101
6.2. Unsuccessful attempts to incorporate NH2Y into alpha2 101
6.3. Successful incorporation of NH2Y into alpha2 103
6.4. Protocol for successful expression of NH2Y-alpha2s 103
6.5. Results 104
6.6. Protocol for purification of NH2Y-alpha2s 104
6.7. Assessment of the fidelity and specificity of NH2Y insertion into alpha2 105
6.8. Protocol for measurement of catalytic activities of NH2Y-alpha2s 107
6.9. Protocol for use of the mechanism-based RNR inhibitor, N3ADP 107
6.10. Results of activity and N3ADP assays 108
7. Characterization of NH2Y-alpha2s 109
Overview 109
Protocol for determining the EPR and UV-vis properties of NH2Y.-alpha2s 110
Results 110
Protocol for determining the kinetics of NH2Y.-alpha2 formation by SF UV-vis spectroscopy 112
Protocol for determining the kinetics of NH2Y.-alpha2 formation by RFQ EPR spectroscopy 112
Results of SF UV-vis and RFQ EPR spectroscopic studies 113
8. Summary 115
Acknowledgments 115
References 115
Chapter 4: Semisynthesis of Proteins Using Split Inteins 121
1. Introduction 122
2. Protein Splicing in cis and in trans is Performed by Inteins 125
3. Design of Split Inteins and Considerations on the Intein Insertion Site 126
4. Materials and Methods 131
4.1. Solid-phase peptide synthesis of ExN-IntN peptides 131
4.2. Construction of IntC-POI plasmids 132
4.3. Expression and purification of IntC-POI fusion proteins 132
4.4. Protein trans-splicing assays 133
4.5. Interaction studies of the IntN/IntC association 134
4.6. Purification and functional analysis of semisynthetic splice products 135
4.7. Protein splicing in complex mixtures 135
5. Summary and Conclusion 136
Acknowledgments 137
References 137
Chapter 5: Expressed Protein Ligation for Metalloprotein Design and Engineering 141
1. Introduction 160
2. Methods of Selenocysteine Incorporation 163
3. Incorporation of Selenocysteine into the Type 1 Copper Site of Azurin 164
3.1. Synthesis of Fmoc-Sec(PMB)-OH 165
3.2. Solid-phase peptide synthesis (SPPS) of C-terminal 17-mer peptide 166
3.3. Deprotection of selenocysteine-containing 17-mer peptide 167
3.4. General procedure for EPL of Azurin(1-111)- Intein-CBD and the C-terminal 17-mer peptide 169
3.5. Characterization of selenocysteine azurin 170
4. Tuning the Type 1 Copper Reduction Potential Using Isostructural Methionine Analogues 171
4.1. Use of EPL for incorporation of methionine analogues 171
4.2. General procedure for the SPPS of methionine analogue 17-mer peptides 171
4.3. General procedure for the peptide cleavage from the resin 172
4.4. General procedure for EPL of azurin(1-111)-intein-CBD and the C-terminal peptides containing methionie analogues 172
4.5. Characterization of methionine analogues of azurin 173
5. Conclusion 173
Acknowledgments 174
References 175
Chapter 6: Using Expressed Protein Ligation to Probe the Substrate Specificity of Lantibiotic Synthetases 142
1. Introduction 143
2. Use of Expressed Protein Ligation to Prepare Substrate Analogues 145
2.1. Overview 145
2.2. General procedure for solid-phase peptide synthesis 148
2.3. General procedure for purification of the peptide thioester His-LctA (1-37)-MES 149
2.4. General procedure for ligation of LctA(1-37)MES with short peptides 150
2.5. Investigation of the dehydration reaction 151
2.6. General procedure for LctM dehydration assays with truncated LctA analogues 152
2.7. Procedure for LctM-catalyzed dehydration of LctA(1-43) with difluoromethylthreonine (8) at position 42 153
2.8. Investigation of the cyclization reaction 153
2.9. General procedure for LctM and LctM-C781A assays with truncated LctA analogues and analysis of the assay products with CNBr 155
3. Conclusion 157
Acknowledgments 158
References 158
Chapter 7: Semisynthesis of K+ Channels 197
1. Introduction 198
2. Experimental Protocols 201
2.1. Synthetic design 201
2.2. Synthesis of the KcsA 70-123 (pF-Phe103) C-peptide 202
2.3. Generation of the N-peptide thioester 204
2.4. The ligation reaction 207
2.5. Folding of the semisynthetic KcsA channel 207
2.6. Functional characterization of the semisynthetic KcsA 208
3. Application of Semisynthesis in Investigating the Selectivity Filter of the K+ Channels 209
3.1. D-Ala substitution in the selectivity filter (Valiyaveetil et al., 2004, 2006) 209
3.2. Ester substitution in the selectivity filter (Valiyaveetil et al., 2006) 210
4. Summary 210
Acknowledgments 211
References 211
Chapter 8: Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance 213
1. Introduction 214
2. Segmental Isotopic Labeling using Expressed Protein Ligation 215
2.1. Overview 215
2.2. Ligation site 217
2.3. Synthesis of a segment with C-terminal alpha- thioester 220
2.4. Synthesis of a segment with N-terminal cysteine 220
2.5. Ligation protocol 221
3. Segmental Labeling using Protein Trans-Splicing 221
4. Multiple Segment Assembly 222
5. Segmental Labeling of C-Terminal SRC Kinase(Csk) 222
5.1. Overview 222
5.2. Cloning Csk SH32 and kinase gene to expression vector 225
5.3. Expression and purification Csk SH32 with C-terminal Mxe GyrA intein (intein2) 226
5.4. Expression and purification of Csk kinase with N-terminal Ssp DnaB intein (intein1) 227
5.5. Ligation of SH32 domain with testing peptide 229
5.6. Ligation of SH32 with kinase domain 230
5.7. Purification of ligation product 230
5.8. NMR spectroscopy 232
Acknowledgments 233
References 233
Chapter 9: Semisynthesis of Membrane-Attached Prion Proteins 239
1. Introduction 240
2. Chemical Synthesis of Membrane Anchors 241
3. Semisynthesis of rPrPPalm by Expressed Protein Ligation 242
3.1. Cloning procedure 243
3.2. Bacterial expression and protein purification 244
3.3. Intein cleavage and purification 244
3.4. Native chemical ligation reactions 245
3.5. Folding of rPrPPalm and rPrPGPI 246
3.6. Comments 247
4. Semisynthesis of rPrPPalm by Protein Trans-Splicing 247
4.1. Cloning procedure 247
4.2. Bacterial expression and protein purification 247
4.3. Refolding of rPrP-DnaEN-His 248
4.4. Synthesis of DnaEC-membrane anchor peptides 248
4.5. Trans-splicing with rPrP-DnaEN-His 249
4.6. Folding of rPrPPalm 249
4.7. Comments 250
5. Liposome Attachment and Aggregation of rPrPPalm and rPrPGPI 250
5.1. Vesicle attachment of rPrPPalm and rPrPGPI 251
5.2. Aggregation assays 251
6. Conclusion 252
References 252
Chapter 10: Use of Intein-Mediated Protein Ligation Strategies for the Fabrication of Functional Protein Arrays 257
1. Introduction 258
2. Intein-Mediated Protein Ligation Strategies 259
2.1. N-terminal intein fusions for protein immobilization 261
2.2. C-terminal intein fusions for protein biotinylation and immobilization 264
3. In Vitro, In Vivo and Cell Free Strategies for Protein Biotinylation at the C-Terminal 265
4. Methods 269
4.1. Protocols for biotinylation of proteins at the C-terminus 269
4.1.1. In vitro protein biotinylation 269
4.1.1.1. Practical considerations 271
4.1.2. In vivo protein biotinylation 277
4.1.3. Cell-free protein expression and biotinylation 277
4.2. Protocol for generation of N-terminal cysteine-containing proteins 278
4.3. Preparation of slides 279
4.3.1. Thioester slides 280
4.3.2. Avidin slides 280
4.4. Spotting of slides and detection of proteins 280
5. Concluding Remarks 281
References 282
Chapter 11: Semisynthesis of Ubiquitylated Proteins 287
1. Introduction 288
2. Semisynthesis of Ubiquitylated Histone H2B 290
2.1. Overall synthetic design 290
3. Methods 292
3.1. General methods 292
4. Synthesis of Photocleavable Ligation Auxiliary 293
4.1. 4-(2-Methoxy-5-nitro-4-vinyl-phenoxy)-butyric acid methyl ester (2) 293
4.2. 4-[4-(1-tert-Butoxycarbonylamino-2-hydroxy-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid methyl easter (3) 294
4.3. 4-[4-(2-Acetylsulfanyl-1-tert-butoxycarbonylamino-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid mehyl ester (4) 295
4.4. 4-[4-(1-tert-Butoxycarbonylamino-2-tert-butyldisulfanyl-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid (5) 295
4.5. 4-[4-(1-Amino-2-tert-butyldisulfanyl-ethyl)-2-methoxy-5-nitro-phenoxy]-N-methyl-butyramide (6) 296
5. Peptide Synthesis 296
5.1. Chemical synthesis of peptide 7 297
6. Generation of Recombinant Protein alpha- Thioesters 297
6.1. Preparation of ubiquitin(1-75)- alpha- MES (8) 298
6.2. Preparation of H2B(1-116)-alpha- MES (9) 298
7. Expressed Protein Ligation 299
7.1. Synthesis of ubiquitylated peptide 10 300
7.2. Photolytic deprotection of 10 to give branched protein 11 300
7.3. Synthesis of ubiquitylated H2B mutant 12 301
7.4. Desulfurization of 12 to give uH2B 13 301
8. Generation of Ubiquitylated Mononucleosomes 301
8.1. Recombinant histone preparation 302
8.2. Preparation of DNA for nucleosome formation 303
8.3. Ubiquitylated nucleosome formation 303
9. Conclusions 304
Acknowledgments 304
References 304
Author Index 307
Subject Index 321
Color Plate 327