Integrated Methods in Protein Biochemistry: Part A

1st Edition, Volume 675 - October 9, 2022
Imprint: Academic Press
Editor: Arun K. Shukla
Language: English
Hardback ISBN:
9 7 8 - 0 - 3 2 3 - 9 9 2 6 6 - 4
eBook ISBN:
9 7 8 - 0 - 3 2 3 - 9 9 2 6 7 - 1

Integrated Methods in Protein Biochemistry: Part A, Volume 677, the latest release in the Methods in Enzymology series, highlights new advances in the field with this new volume pr… Read more

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Integrated Methods in Protein Biochemistry: Part A, Volume 677, the latest release in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters on topics such as DNA and protein engineering to create protein bioswitches with new functions, Interaction and cross-talk of prelamin A with integral membrane zinc metalloproteases, An experimental protocol to study lipid transfer proteins, Synthesis of small heat shock proteins, Druggable p-p interacting sites for Co-chaperone DNAJA1 and its partner proteins, An experimental protocol for glycoconjugate analysis, Methods for proximity-based biotinylation combined with Mass Spectrometry, and more.

Additional chapters cover Synthetic antibody fragments as conformational sensors of protein activation and trafficking, Expression, purification, functional analysis and crystallization of Rag GTPase, Purification of bacterial transcription elongation complexes by photoreversible immobilization, Inhibition of c-Myc-MAX heterodimerization, Fluorogenic RNA aptamers to probe transcription by multi-subunit RNA polymerases, and much more.

Cover image
Title page
Table of Contents
Copyright
Contributors
Preface
Chapter One: Engineering protein and DNA tools for creating DNA-dependent protein switches
Abstract
1: Introduction
2: Design and cloning of AFF biosensors
3: Design and validation of DNA tools
4: Luminescence activity assays
5: Summary and conclusions
Acknowledgments
References
Chapter Two: Reconstitution and biochemical studies of extended synaptotagmin-mediated lipid transport
Abstract
1: Introduction
2: Molecular cloning and mutation
3: Protein expression and purification
4: Lipid transfer assay
5: Liposome tethering assay
6: Liposome co-flotation assay
7: Lipid transfer direction assay
Acknowledgements
References
Chapter Three: Synthesis of O-GlcNAcylated small heat shock proteins
Abstract
1: Introduction
2: General methods
3: Solid-phase peptide synthesis (SPPS)
4: Expression of protein thioesters
5: Cysteine protection and protein semisynthesis
6: Refolding
7: Summary and conclusions
References
Chapter Four: Druggable sites/pockets of the p53-DNAJA1 protein–protein interaction: In silico modeling and in vitro/in vivo validation
Abstract
1: Introduction
2: Oncogenic missense mutant p53
3: DNAJA1 is a potential key co-chaperone that stabilizes oncogenic mutant p53
4: In silico framework for identifying the druggable and salient protein–protein interacting sites of mutant p53 and DNAJA1 proteins
5: Demonstration of the biologic function of the protein–protein interacting site of DNAJA1 and MutP53R175H on Mutp53R175H stability by molecular biology approaches
6: Applying the machine-learning-based classifier to screen and identify the small molecule inhibitor against the druggable protein–protein interacting site of DNAJA1 and mutant p53
7: In vitro screening of our top hits against the DNAJA1–mutP53R175H interacting pocket on degrading mutP53R175H protein
8: In vitro and in vivo validation of small molecule inhibitor GY1-22 against the druggable protein–protein interacting site of DNAJA1 and mutant p53 and cancer growth
9: Summary
Conflict of interest
References
Chapter Five: RAPIDS, a method for sub-compartmental identification of protein interactomes
Abstract
1: Introduction
2: Before you begin
3: Key resources table
4: Materials and equipment
5: RAPIDS
6: Expected outcomes
7: Statistical analysis
8: Advantages
9: Limitations
10: Optimization and troubleshooting
References
Chapter Six: Purification and biochemical characterization of the Rag GTPase heterodimer
Abstract
1: Introduction
2: Purification of the RagA-RagC heterodimer
3: Nucleotide binding and kinetics
4: GTP hydrolysis kinetics
5: Summary and conclusions
Acknowledgments
References
Chapter Seven: Isolation of synchronized E. coli elongation complexes for solid-phase and solution-based in vitro transcription assays
Abstract
1: Introduction
2: Design and preparation of template DNA
3: Equilibration of streptavidin-coated magnetic beads
4: Isolation of C3-SC1TECs from an in vitro transcription reaction
5: Quality control of C3-SC1TEC activity by time course assay
6: Quality control of C3-SC1TEC purity by electrophoretic mobility shift assay
7: Quality control of C3-SC1TEC yield by intermediate fraction analysis
References
Chapter Eight: Cellular experiments to study the inhibition of c-Myc/MAX heterodimerization
Abstract
1: Introduction
2: Materials and methods
Acknowledgment
References
Chapter Nine: Fluorogenic RNA aptamers to probe transcription initiation and co-transcriptional RNA folding by multi-subunit RNA polymerases
Abstract
1: Introduction
2: Multi-round broccoli-FLAP assay for monitoring transcription initiation
3: Single-round iSpinach-FLAP assay for monitoring co-transcriptional RNA folding
4: Summary and conclusions
Acknowledgments
References
Chapter Ten: Phosphorylation and subcellular localization of human phospholipase A1, DDHD1/PA-PLA1
Abstract
1: Introduction
2: Molecular design of the analysis for the phosphorylation of DDHD1
3: Phospho-proteomics analysis of human DDHD1
4: Site-directed mutagenesis of human DDHD1
5: Phos-tag SDS-PAGE
6: Measurement of phospholipase A1 activity
7: In vitro kinase assay for the identification of responsible protein kinases
8: Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization
9: Summary and conclusions
Acknowledgments
References
Chapter Eleven: Elucidation of folding pathways of knotted proteins
Abstract
1: Introduction
2: Recombinant protein expression and purification
3: Equilibrium unfolding
4: Protein folding kinetics
5: Other methods
6: Summary and discussion
Acknowledgment
References
Chapter Twelve: Biophysical and structural characterizations of the effects of mutations on the structure–activity relationships of SARS-CoV-2 spike protein
Abstract
1: Introduction
2: Key resources table
3: Recombinant protein production
4: Thermal stability analyses of S protein variants
5: Folding stability analyses of S protein variants by NSEM
6: Kinetic analysis of receptor and antibody binding to S protein variants
7: Structure–activity relationship analysis of s protein variants by cryo-EM
8: Concluding remarks
Acknowledgments
References
Chapter Thirteen: A gel electrophoresis-based assay for measuring enzymatic RNA decapping activity
Abstract
1: Introduction
2: Safety measures
3: Materials and reagents
4: In vitro transcription
5: Enzymatic dose curve assay
6: Gel electrophoresis and Imaging
7: Analysis
Acknowledgments
References
Chapter Fourteen: Nanoluciferase-based cell fusion assay for rapid and high-throughput assessment of SARS-CoV-2-neutralizing antibodies in patient samples
Abstract
1: Introduction
2: Before you begin
3: Materials and equipment
4: Step-by-step method details
5: Expected outcome and quantification
6: Advantages
7: Limitations
8: Alternative methods/procedures
Acknowledgments
References
Chapter Fifteen: Linchpin-directed precise labeling of lysine in native proteins, purification, and analysis
Abstract
1: Introduction
2: Linchpin-directed modification (LDM®)
3: Materials
4: Procedure
5: Analysis
6: Prospects and conclusion
Acknowledgments
References
Chapter Sixteen: In vitro and in cellulae methods for determining the target protein SUMOylation
Abstract
1: Introduction
2: General methods of plasmid isolation, protein expression and purification
3: SUMOylation assays
4: Materials and equipment
5: Step-by-step protocols
6: Summary and conclusions
Acknowledgments
References
Chapter Seventeen: Two-dimensional electrophoresis–cellular thermal shift assay (2DE-CETSA) for target identification of bioactive compounds
Abstract
1: Introduction
2: General methods and cell culture
3: Sample preparation for 2DE-CETSA
4: 2DE and data acquisition
5: Data processing and protein identification
6: Summary and conclusions
Acknowledgments
References

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