Modifications and targeting of protein termini. Part A. /

Modifications and Targeting of Protein Termini, Volume 684 in the Methods in Enzymology series serial highlights new advances in the field, with this new volume presenting interesting chapters pn a variety of timely topics, including Optimizing purification and activity assays of N-terminal methyltr...

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Bibliographic Details
Main Author:	Arnesen, Thomas (Author)
Corporate Author:	ScienceDirect (Online service)
Format:	eBook
Language:	English
Published:	Kidlington, England : Academic Press, [2023]
Series:	Issn Series.
Subjects:	Proteins > Analysis. Life sciences. Protéines > Analyse. Sciences de la vie. biological sciences. Electronic books.
Online Access:	Connect to the full text of this electronic book

Table of Contents:

Intro
Modifications and Targeting of Protein Termini: Part A
Copyright
Contents
Contributors
Preface: A nascent polypeptide emerges
Chapter One: Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells
1. Introduction
2. Experimental design
3. General considerations
4. Cell growth, in vivo crosslinking, and cell lysis
4.1. Equipment, materials, and buffer recipes
4.2. Cell growth
4.3. In vivo crosslinking (optional)
4.4. Cell lysis
5. Nuclease digestion and polysome profiling
5.1. Equipment for polysome profiling
5.2. Materials and buffer recipes
5.3. Nuclease digest
5.4. Polysome profiling
6. Ribosome isolation for total translatome and selected translatome
6.1. Ribosome isolation by sucrose cushion centrifugation
6.1.1. Equipment
6.1.2. Materials and buffer recipes
6.1.3. Procedure
6.2. Factor-bound RNC isolation from purified ribosomes
6.2.1. Equipment
6.2.2. Materials and buffer recipes
6.2.3. Procedure
6.3. Factor-bound RNC isolation from cell lysate
6.3.1. Equipment
6.3.2. Materials and buffer recipes
6.3.3. Procedure
7. Preparation of a ribosome footprint library
7.1. Equipment
7.2. Materials and buffer recipes
7.3. Oligonucleotides
7.4. General methods
7.4.1. Precipitation of RNA
7.4.2. Precipitation of DNA
7.4.3. Polyacrylamide gel electrophoresis and gel extraction of nucleic acids
7.5. Phenol-chloroform extraction
7.6. Gel purification of ribosome-protected footprints
7.7. Dephosphorylation
7.8. Quantification of RNA and rRNA depletion
7.9. Linker ligation at 3 end
7.10. Reverse transcription of 3 ligated footprints to ssDNA
7.11. Circularization of ssDNA
7.12. PCR amplification
7.13. Library quality control and deep sequencing.
8. Data analysis
9. Prospects and conclusion
Acknowledgments
References
Chapter Two: Deformylation of nascent peptide chains on the ribosome
1. Introduction
2. General methods
3. PDF purification
3.1. Purification of His6-tagged PDF
3.1.1. Equipment
3.1.2. Buffers and reagents
3.1.3. Procedure
3.1.4. Notes
3.2. Preparation of fluorescent PDF(Bpy)
3.2.1. Equipment
3.2.2. Buffers and reagents
3.2.3. Procedure
3.2.4. Notes
4. PDF substrates
5. RNC deformylation
5.1. Steady-state deformylation kinetics
5.1.1. Equipment
5.1.2. Buffer and reagents
5.1.3. Procedure
5.1.4. Analysis
5.1.5. Notes
5.2. Pre-steady-state kinetics
5.2.1. Equipment
5.2.2. Buffer and reagents
5.2.3. Procedure
5.2.4. Analysis
5.2.5. Notes
6. PDF-ribosome interactions
6.1. PDF binding to the ribosome
6.1.1. Equipment
6.1.2. Buffer and reagents
6.1.3. Procedure
6.1.4. Analysis
6.1.5. Notes
6.2. Pre-steady-state kinetics
6.2.1. Equipment
6.2.2. Buffer and reagents
6.2.3. Procedure
6.2.4. Analysis
6.2.5. Notes
7. Conclusion
Acknowledgments
References
Chapter Three: Optimizing purification and activity assays of N-terminal methyltransferase complexes
1. Introduction
2. Design of single and dual expression constructs
2.1. Equipment
2.2. Reagents
2.3. Procedure
2.4. Notes
3. Purification of recombinant protein
3.1. Equipment
3.2. Buffers, strains, and reagents
3.3. Procedure
3.4. Notes
4. Western blot in vitro methyltransferase assays for full-length protein substrates
4.1. Equipment
4.2. Buffers, strains, and reagents
4.3. Procedure
4.4. Data analysis
4.5. Notes
5. Luminescent in vitro methyltransferase assays for peptide substrates
5.1. Equipment
5.2. Buffers and reagents.
2.5.2. For storage at -20C
3. Preparation of noncommercial N-myristoyl protein or CoA derivatives
3.1. Myristoyl-CoA and any unusual CoA acyl derivatives
3.2. Myristoylated protein substrates
3.2.1. Fatty acid tagging of target proteins with NMT in vitro
3.2.2. Myristoyl tagging in cell-free translation systems
3.2.3. Bacteria-driven target labeling
3.2.4. Quality controls
4. Characterization of NMT-induced modifications on protein targets
4.1. General considerations
4.2. MALDI mass spectrometry
4.2.1. Main outlines
4.2.2. Notes
4.3. The NMT/IpaJ pipeline
4.4. Click chemistry-based protein imaging
4.4.1. Overview
4.4.2. In cellulo labeling procedures with acyl precursors
4.4.3. In-gel imaging
4.4.4. Immunoprecipitation
4.4.5. Fluorescence imaging after immunoblotting
5. Structural studies of NMT and its complexes with substrates and products
5.1. Structural overview
5.2. Crystallization and structure determination
5.2.1. General crystallization conditions
5.2.2. Obtaining crystals of NMT in complex with reactants or reaction intermediates
5.2.3. Dataset collection, structure resolution, and refinement
6. Conclusions and future prospects
Acknowledgments
Funding
References
Chapter Six: Kinetic and catalytic features of N-myristoyltransferases
1. Introduction
2. Materials, reagents, and buffers
2.1. Equipment
2.2. Enzymes
2.3. Reagents
2.3.1. Fatty acyl derivatives
2.3.2. Peptides
2.3.3. Notes
2.4. Buffer components
2.4.1. For storage at room temperature
2.4.2. For storage at 4C
2.4.3. For storage at -20C
3. Kinetic analysis
3.1. GNAT-specific kinetic properties and considerations
3.2. Discontinuous assays
3.2.1. Radioactive labeling
3.2.2. HPLC assays
3.3. Continuous assays.
3.3.1. NMT activity coupling conditions
3.3.2. Acylation kinetics
3.3.3. Notes
3.4. Catalytic efficiencies of various substrates: Ranges and meanings
3.4.1. CoA donors
3.4.2. Polypeptide acceptor donor
4. Conclusions
Acknowledgments
Funding
References
Chapter Seven: Use of alkyne-tagged myristic acid to detect N-terminal myristoylation
1. Introduction
2. Synthesis of Alk-12probes
2.1. 2-Tetradecyn-1-ol
2.2. 13-Tetradecyn-1-ol
2.3. 13-Tetradecynoic acid (Alk-12)
3. Alk-12 and rhodamine labeling of targeted proteins in cells with click chemistry
3.1. Equipment
3.2. Materials, buffers, and reagents
3.3. Procedure
3.4. Notes
4. Alk-12 and PEG-5000 labeling of targeted proteins
4.1. Equipment
4.2. Materials, buffers, and reagents
4.3. Procedure
4.4. Notes
5. Global labeling of N-myristoylated proteins in whole cell lysate
5.1. Equipment
5.2. Materials, buffers, and reagents
5.3. Procedure
5.4. Notes
6. Global profiling of N-myristoylated proteins using SILAC proteomics
6.1. Equipment
6.2. Materials, buffers, and reagents
6.3. Procedure
6.4. Notes
References
Chapter Eight: Peptide CoA conjugates for in situ proteomics profiling of acetyltransferase activities
1. Introduction
2. Peptide probe synthesis
2.1. Before you begin
2.2. Key resources table
2.3. Materials and equipment
2.4. Step-by-step method details
2.4.1. Automated solid-phase peptide synthesis (SPPS)
2.4.2. Peptide modifications
2.4.3. Peptide cleavage and purification
2.4.4. CoA conjugation
2.4.5. Copper-catalyzed alkyne-azide cycloaddition (CuAAC)
2.5. Expected outcomes
2.6. Advantages
2.7. Limitations
2.8. Optimization and troubleshooting
2.9. Safety considerations and standards
2.10. Alternative methods/procedures.