Non-Natural Amino Acids

Title Page
......Page 3
Copyright Page.pdf......Page 4
Table of Contents.pdf......Page 5
Contributors to Volume 462.pdf......Page 9
Preface.pdf......Page 12
Methods in Enzymology.pdf......Page 15
Protein 
Phosphorylation by Semisynthesis: From Paper to Practice......Page 42
Overview of Protein Phosphorylation......Page 43
Investigating Protein Phosphorylation with Phosphomimetics......Page 44
Incorporation of alternative genetically encoded phosphomimetics......Page 45
Phosphonate Analogues and Protein Semisynthesis......Page 46
Methods......Page 47
Choosing the peptide ligation site......Page 49
Design of recombinant constructs for semisynthesis......Page 50
Peptide synthesis......Page 51
C-terminal semisynthesis (EPL) on a soluble protein......Page 52
C-terminal EPL on an insoluble protein......Page 54
Kinetic analysis of phosphonylated enzymes......Page 55
Microinjection of phosphonylated enzymes......Page 57
Future of Protein Semisynthesis in Signaling......Page 60
References......Page 62
Protein 
Engineering with the Traceless Staudinger Ligation......Page 66
Traceless Staudinger Ligation......Page 67
Choice of Coupling Reagent......Page 69
Experimental procedure: Synthesis of phosphinothiol I......Page 72
Preparation of the Azido Fragment......Page 74
Experimental procedure: Strategy N1......Page 75
Preparation of the Phosphinothioester Fragment......Page 76
Experimental procedure: Strategy C1......Page 79
Experimental procedure: Strategy C5......Page 80
Experimental procedure: Traceless Staudinger ligation on a solid phase......Page 81
Prospectus......Page 82
Experimental procedure: General......Page 83
References......Page 84
Replacement of Y730 and Y731 in the alpha2 Subunit of Escherichia coli Ribonucleotide Reductase with 3-Aminotyrosine using an Evolved Suppressor tRNA/tRNA-Synthetase Pair
......Page 86
Introduction......Page 88
Site-Specific Insertion of Unnatural Amino Acids using the Suppressor tRNA/RS Method......Page 90
Protocol for assessing uptake and toxicity of NH2Y in E. coli
......Page 92
Results......Page 93
Directed Evolution of NH2Y-RS in E. coli......Page 94
Protocol for incorporation of NH2Y into the Z-domain......Page 96
Results......Page 97
Unsuccessful attempts to incorporate NH2Y into ?2......Page 98
Protocol for successful expression of NH2Y-?2s......Page 100
Protocol for purification of NH2Y-?2s......Page 101
Assessment of the fidelity and specificity of NH2Y insertion into ?2......Page 102
Protocol for use of the mechanism-based RNR inhibitor, N3ADP......Page 104
Results of activity and N3ADP assays......Page 105
Overview......Page 106
Results......Page 107
Protocol for determining the kinetics of NH2Y.-?2 formation by RFQ EPR spectroscopy......Page 109
Results of SF UV-vis and RFQ EPR spectroscopic studies......Page 110
References......Page 112
Semisynthesis of Proteins Using Split Inteins
......Page 118
Introduction......Page 119
Protein Splicing in cis and in trans is Performed by Inteins......Page 122
Design of Split Inteins and Considerations on the Intein Insertion Site......Page 123
Solid-phase peptide synthesis of ExN-IntN peptides......Page 128
Expression and purification of IntC-POI fusion proteins......Page 129
Protein trans-splicing assays......Page 130
Interaction studies of the IntN/IntC association......Page 131
Protein splicing in complex mixtures......Page 132
Summary and Conclusion......Page 133
References......Page 134
Expressed 
Protein Ligation for Metalloprotein Design and Engineering......Page 138
Introduction......Page 139
Methods of Selenocysteine Incorporation......Page 142
Incorporation of Selenocysteine into the Type 1 Copper Site of Azurin......Page 143
Synthesis of Fmoc-Sec(PMB)-OH......Page 144
Solid-phase peptide synthesis (SPPS) of C-terminal 17-mer peptide......Page 145
Deprotection of selenocysteine-containing 17-mer peptide......Page 146
General procedure for EPL of Azurin(1-111)- Intein-CBD and the C-terminal 17-mer pept......Page 148
Characterization of selenocysteine azurin......Page 149
General procedure for the SPPS of methionine analogue 17-mer peptides......Page 150
General procedure for EPL of azurin(1-111)-intein-CBD and the C-terminal peptides containing me......Page 151
Conclusion......Page 152
Acknowledgments......Page 153
References......Page 154
Using Expressed Protein Ligation to Probe the Substrate Specificity of Lantibiotic Synthetases......Page 157
Introduction......Page 158
Overview......Page 160
General procedure for solid-phase peptide synthesis......Page 163
General procedure for purification of the peptide thioester His-LctA (1-37)-MES......Page 164
General procedure for ligation of LctA(1-37)MES with short peptides......Page 165
Investigation of the dehydration reaction......Page 166
General procedure for LctM dehydration assays with truncated LctA analogues......Page 167
Investigation of the cyclization reaction......Page 168
General procedure for LctM and LctM-C781A assays with truncated LctA analogues and analysis of the assay products with......Page 170
Conclusion......Page 172
References......Page 173
Semisynthesis of Kplus Channels
......Page 175
Introduction......Page 176
Synthetic design......Page 179
Synthesis of the KcsA 70-123 (pF-Phe103) C-peptide
......Page 180
Generation of the N-peptide thioester......Page 182
Folding of the semisynthetic KcsA channel......Page 185
Functional characterization of the semisynthetic KcsA......Page 186
D-Ala substitution in the selectivity filter (Valiyaveetil et al., 2004, 2006)......Page 187
Summary......Page 188
References......Page 189
Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance
......Page 191
Introduction......Page 192
Overview......Page 193
Ligation site......Page 195
Synthesis of a segment with N-terminal cysteine......Page 198
Segmental Labeling using Protein Trans-Splicing......Page 199
Overview......Page 200
Cloning Csk SH32 and kinase gene to expression vector......Page 203
Expression and purification Csk SH32 with C-terminal Mxe GyrA intein (intein2)
......Page 204
Expression and purification of Csk kinase with N-terminal Ssp DnaB intein (intein1)......Page 205
Ligation of SH32 domain with testing peptide......Page 207
Purification of ligation product......Page 208
NMR spectroscopy......Page 210
References......Page 211
Semisynthesis of Membrane-Attached Prion Proteins
......Page 216
Introduction......Page 217
Chemical Synthesis of Membrane Anchors......Page 218
Semisynthesis of rPrPPalm by Expressed Protein Ligation......Page 219
Cloning procedure......Page 220
Intein cleavage and purification......Page 221
Native chemical ligation reactions......Page 222
Folding of rPrPPalm and rPrPGPI......Page 223
Bacterial expression and protein purification......Page 224
Synthesis of DnaEC-membrane anchor peptides
......Page 225
Folding of rPrPPalm......Page 226
Liposome Attachment and Aggregation of rPrPPalm and rPrPGPI......Page 227
Aggregation assays......Page 228
References......Page 229
Use of Intein-Mediated Protein Ligation Strategies for the Fabrication of Functional Protein Arrays
......Page 233
Introduction......Page 234
Intein-Mediated Protein Ligation Strategies......Page 235
N-terminal intein fusions for protein immobilization......Page 237
C-terminal intein fusions for protein biotinylation and immobilization......Page 240
In Vitro, In Vivo and Cell Free Strategies for Protein Biotinylation at the C-Terminal......Page 241
In vitro protein biotinylation......Page 245
Practical considerations......Page 247
Cell-free protein expression and biotinylation......Page 253
Protocol for generation of N-terminal cysteine-containing proteins......Page 254
Preparation of slides......Page 255
Spotting of slides and detection of proteins......Page 256
Concluding Remarks......Page 257
References......Page 258
Semisynthesis of Ubiquitylated Proteins......Page 262
Introduction......Page 263
Overall synthetic design......Page 265
General methods......Page 267
4-(2-Methoxy-5-nitro-4-vinyl-phenoxy)-butyric acid methyl ester (2)......Page 268
4-[4-(1-tert-Butoxycarbonylamino-2-hydroxy-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid methyl ester (3)......Page 269
4-[4-(1-tert-Butoxycarbonylamino-2-tert-butyldisulfanyl-ethyl)-2-methoxy-5-nitro-phenoxy]-butyric acid (5)......Page 270
Peptide Synthesis......Page 271
Generation of Recombinant Protein alpha-Thioesters......Page 272
Preparation of H2B(1-116)-alpha-MES (9)......Page 273
Expressed Protein Ligation......Page 274
Photolytic deprotection of 10 to give branched protein 11......Page 275
Generation of Ubiquitylated Mononucleosomes......Page 276
Recombinant histone preparation......Page 277
Ubiquitylated nucleosome formation......Page 278
References......Page 279
Author Index.pdf......Page 281
Subject Index.pdf......Page 294