Protein termini represent a major route to protein regulation. From the moment the very first amino acid of a polypeptide chain exits the ribosome there is potential for steering from the cellular environment. This volume of Methods in Enzymology Modifications and Targeting of Protein Termini focuses on Protein N-termini and C-termini and their modifications which include acetylation, arginylation, myristoylation and oxidation. Also, the impact of terminal modifications is covered, in particular the impact on protein turnover and the ubiquitin E3 ligases which specifically recognize protein N-termini (N-degrons) and C-termini (C-degrons). In addition to the detailed methods and laboratory protocols, the chapters include informative overviews and reviews of the different subfields.
1. Cover image - p. i 2. Title page - p. ii 3. Table of Contents - p. iii 4. Series Page - p. iii 5. Copyright - p. iii 6. Contributors - p. xiii 7. Chapter One: Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases - p. 1 8. Chapter Two: Purification and activity assays of N-terminal acetyltransferase D - p. 19 9. Chapter Three: A fluorescent CPM-based in vitro acetylation assay: A tool for assessing N-terminal acetyltransferase activity and profiling compound activity - p. 51 10. Chapter Four: In vitro acetyltransferase activity assays for N-terminal acetyltransferases - p. 87 11. Chapter Five: Parallel reaction monitoring reveals N-terminal acetylation of plastid precursor proteins - p. 107 12. Chapter Six: N-terminal acetylation-specific antibodies: Specificity determination by mass spectrometry and utilization in in vitro acetylation assays - p. 129 13. Chapter Seven: Functional assessment of N-terminal acetyltransferase variants - p. 167 14. Chapter Eight: A pan-N-formylmethionine-specific antibody as a tool for analyzing Nα-terminal formylation - p. 187 15. Chapter Nine: Chemical proteomic approaches to investigate N-myristoylation - p. 217 16. Chapter Ten: Proteome-wide analysis of the oxidation state of initiator methionines - p. 244 17. Chapter Eleven: A biophysical approach to studying N-terminal cysteine oxidase substrate preferences - p. 259 18. Chapter Twelve: Recombinant expression, purification, and characterization of human ATE1 arginyltransferase - p. 285 19. Chapter Thirteen: In-bacteria arginylation assay - p. 297 20. Chapter Fourteen: Identification of arginylated N-termini with specific reagents - p. 309
Protein termini represent a major route to protein regulation. From the moment the very first amino acid of a polypeptide chain exits the ribosome there is potential for steering from the cellular environment. This volume of Methods in Enzymology Modifications and Targeting of Protein Termini focuses on Protein N-termini and C-termini and their modifications which include acetylation, arginylation, myristoylation and oxidation. Also, the impact of terminal modifications is covered, in particular the impact on protein turnover and the ubiquitin E3 ligases which specifically recognize protein N-termini (N-degrons) and C-termini (C-degrons). In addition to the detailed methods and laboratory protocols, the chapters include informative overviews and reviews of the different subfields.
1. Cover image - p. i 2. Title page - p. ii 3. Table of Contents - p. iii 4. Series Page - p. iii 5. Copyright - p. iii 6. Contributors - p. xiii 7. Chapter One: Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases - p. 1 8. Chapter Two: Purification and activity assays of N-terminal acetyltransferase D - p. 19 9. Chapter Three: A fluorescent CPM-based in vitro acetylation assay: A tool for assessing N-terminal acetyltransferase activity and profiling compound activity - p. 51 10. Chapter Four: In vitro acetyltransferase activity assays for N-terminal acetyltransferases - p. 87 11. Chapter Five: Parallel reaction monitoring reveals N-terminal acetylation of plastid precursor proteins - p. 107 12. Chapter Six: N-terminal acetylation-specific antibodies: Specificity determination by mass spectrometry and utilization in in vitro acetylation assays - p. 129 13. Chapter Seven: Functional assessment of N-terminal acetyltransferase variants - p. 167 14. Chapter Eight: A pan-N-formylmethionine-specific antibody as a tool for analyzing Nα-terminal formylation - p. 187 15. Chapter Nine: Chemical proteomic approaches to investigate N-myristoylation - p. 217 16. Chapter Ten: Proteome-wide analysis of the oxidation state of initiator methionines - p. 244 17. Chapter Eleven: A biophysical approach to studying N-terminal cysteine oxidase substrate preferences - p. 259 18. Chapter Twelve: Recombinant expression, purification, and characterization of human ATE1 arginyltransferase - p. 285 19. Chapter Thirteen: In-bacteria arginylation assay - p. 297 20. Chapter Fourteen: Identification of arginylated N-termini with specific reagents - p. 309
