Non-Natural Amino Acids (Volume 462) (Methods in Enzymology, Volume 462)

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. This volume brings together the tools and principles of chemistry with the molecules and processes of living cells to create molecules with new properties and functions found neither in nature nor in the test tube. By studying the structure and function of these resulting molecules, new insights can be gained into the molecular mechanisms of complex biological and chemical systems. 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. By studying the structure and function of non-natural molecules, new insights can be gained into the molecular mechanisms of complex biological and chemical systems.Provides a "one-stop shop" for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods 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 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 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 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. This volume brings together the tools and principles of chemistry with the molecules and processes of living cells to create molecules with new properties and functions found neither in nature nor in the test tube. By studying the structure and function of these resulting molecules, new insights can be gained into the molecular mechanisms of complex biological and chemical systems. 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. By studying the structure and function of non-natural molecules, new insights can be gained into the molecular mechanisms of complex biological and chemical systems. Provides a "one-stop shop" for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods Front Cover; Methods in Enzymology; Copyright Page; Contents; Contributors; Preface; Methods in Enzymology; Chapter 1: Protein Phosphorylation by Semisynthesis: From Paper to Practice; Chapter 2: Protein Engineering with the Traceless Staudinger Ligation; 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; Chapter 4: Semisynthesis of Proteins Using Split Inteins; Chapter 5: Expressed Protein Ligation for Metalloprotein Design and Engineering