PDZ Mediated Interactions: Methods and Protocols (Methods in Molecular Biology, 2256)

Preface
Contents
Contributors
Chapter 1: Identification of PDZ Interactions by Yeast Two-Hybrid Technique
	1 Introduction
	2 Materials
		2.1 Design of Primers for Bait Constructs
		2.2 PCR Amplification and Purification of the Bait Constructs
		2.3 Cloning of the Bait Constructs
		2.4 Y2H Yeast Strains
		2.5 Yeast Culture Media
		2.6 Yeast Transformation
		2.7 Screening of the Human PDZome Library
	3 Methods
		3.1 Amplification and Purification of the Y2H Bait Constructs
		3.2 Cloning of Bait Entry Clones in pDONr/pZeo
		3.3 Cloning of the Bait Constructs in pGBT9-GW
		3.4 Yeast Transformation of the Bait Constructs
		3.5 Validating the Bait Constructs
		3.6 Yeast Two-Hybrid Screening of PDZome Array
	4 Notes
	References
Chapter 2: Identification of PDZ Interactions by Affinity Purification and Mass Spectrometry Analysis
	1 Introduction
	2 Materials
		2.1 Common Reagents and Buffers
		2.2 Basic Reagents for Immunopurification
		2.3 Basic Reagents for Peptide-Based Purification
		2.4 Sample Digestion
	3 Methods
		3.1 Immunopurification
			3.1.1 Mammalian Expression Vectors
			3.1.2 Verification of Protein Expression by Western Blot
			3.1.3 Mammalian Stable Cell Lines
			3.1.4 Characterization of Stable Cell Lines
			3.1.5 Amplification of Expressing Cells for Large-Scale Purification
			3.1.6 Preparation of Cell Extracts and Immunopurification
			3.1.7 Elution
		3.2 Peptide-Based Purification
			3.2.1 Design of Peptide Containing a PDZBM
			3.2.2 Coupling the Peptide to NHS Beads
			3.2.3 Coupling Biotinylated Peptides with Streptavidin Beads
			3.2.4 Peptide Pulldown
		3.3 Trypsin Digestion of Protein Complexes and Preparation for Mass Spectrometry Analysis
			3.3.1 Eluted Sample Preparation
			3.3.2 Trypsin Digestion
			3.3.3 Mass Spectrometry Analysis
		3.4 Mass Spectrometry-Based Quantification
			3.4.1 Protein Identification and Quantification Using MaxQuant
			3.4.2 Statistical Analysis Using Perseus
		3.5 Results
			3.5.1 Identification of TANC1 Associated Protein Complexes
			3.5.2 Validation of the Newly Identified TANC1-Associated PDZ Proteins
		3.6 Discussion
	4 Notes
	References
Chapter 3: Identification of PDZ Interactions by Proteomic Peptide Phage Display
	1 Introduction
	2 Materials
		2.1 Oligonucleotide Pool Amplification
		2.2 Purification of dU-ssDNA Phagemid
		2.3 In Vitro Synthesis of Phagemid dsDNA Library
		2.4 Electroporation and Amplification of Library
		2.5 Protein Expression and Purification
		2.6 Phage Selections
		2.7 Phage Pool Enzyme Linked Immunosorbent Assay (ELISA)
		2.8 Preparation and Quantification of Sample for Next-Generation Sequencing (NGS)
	3 Methods
		3.1 PCR Amplification of a Commercial Custom Oligo Pools
		3.2 Quantification of the PCR Product (See Note 1)
		3.3 Purification of dU-ssDNA Phagemid
		3.4 In Vitro Synthesis of the Phagemid dsDNA Library
		3.5 Electroporation of Phagemid Library and Amplification of Phage Library
		3.6 Bait Protein Expression and Purification
			3.6.1 Protein Expression
			3.6.2 Batch Purification
		3.7 ProP-PD Selection Against PDZ Domains
			3.7.1 Day 0
			3.7.2 Selection Day 1
			3.7.3 Selection Day 2-4
			3.7.4 Day 5: Phage pool ELISA
		3.8 Preparation of Sample for NSG Analysis
			3.8.1 PCR Amplification and Barcoding
			3.8.2 Normalization of PCR Products
	4 Notes
	References
Chapter 4: A Computational Protocol to Analyze PDZ/PBM Affinity Data Obtained by High-Throughput Holdup Assay
	1 Introduction
	2 Materials
		2.1 Capillary Electrophoresis Instrument´s Software
		2.2 Computer
		2.3 SPIKE Package and Software Availability
	3 Methods
		3.1 Input Data Extraction from the Quantitative Capillary Electrophoresis Instrument
		3.2 Transforming the Input  Data
		3.3 Checking the Data Quality
		3.4 Extracting BIs and Other  Data
		3.5 Storing and Plotting the  Data
	4 Notes
	References
Chapter 5: Study of PDZ-Peptide and PDZ-Lipid Interactions by Surface Plasmon Resonance/BIAcore
	1 Introduction
	2 Materials
		2.1 Lyophilized Synthetic Biotinylated Peptides
		2.2 Liposomes: We Recommend to Use Liposomes Mimicking the Lipid Composition of the Biological Membranes of Interest
		2.3 BIAcore
		2.4 Chips
	3 Methods
		3.1 Immobilizing Biotinylated Compounds on BIAcore SA Sensor  Chip
		3.2 kon/koff Determination Using a Biotinylated Peptide-Loaded BIAcore SA Sensor  Chip
		3.3 Calculate Steady State Affinity in Multicycle Kinetics Analysis
		3.4 Preparation of Large Unilamellar Vesicles (LUVs) by Extrusion
		3.5 Immobilizing LUVs on BIAcore L1 Sensor Chips
		3.6 Start an Experiment in BIAcore with an L1  Chip
		3.7 Study of Tripartite Complexes in BIAcore with a SA Sensor  Chip
	4 Notes
	References
Chapter 6: PDZ Sample Quality Assessment by Biochemical and Biophysical Characterizations
	1 Introduction
		1.1 Purity
			1.1.1 SDS-PAGE Electrophoresis
			1.1.2 Capillary Gel Electrophoresis
			1.1.3 UV-Visible Spectroscopy Between 200 nm and 340 nm
		1.2 Identity
			1.2.1 Intact Mass Spectrometry
			1.2.2 Top-Down PDZ Sequencing
		1.3 Homogeneity
			1.3.1 Dynamic Light Scattering
			1.3.2 Analytical Size Exclusion Chromatography
			1.3.3 Analytical Ultracentrifugation
		1.4 Conformational Stability/Folding State
			1.4.1 Circular Dichroism (CD)
			1.4.2 Differential Scanning Calorimetry (DSC) and Differential Scanning Fluorimetry (DSF)
			1.4.3 Nuclear Magnetic Resonance (NMR)
	2 Materials
		2.1 Purity
			2.1.1 Basic Reagents for SDS-PAGE Electrophoresis
			2.1.2 Capillary Electrophoresis (CE)
			2.1.3 UV Spectroscopy
		2.2 Integrity Measurements by MALDI-TOF
			2.2.1 Total  Mass
			2.2.2 ISD and T3 Sequencing
		2.3 Homogeneity
			2.3.1 Dynamic Light Scattering (DLS)
			2.3.2 Size Exclusion Chromatography (SEC) with Multi Angle Light Scattering (SEC-MALS)
		2.4 Conformational Stability/Folding State
			2.4.1 Circular Dichroism (CD)
			2.4.2 Differential Scanning Fluorimetry (Nano DSF)
			2.4.3 NMR
	3 Methods
		3.1 Purity
			3.1.1 SDS-Page
			3.1.2 Capillary Gel Electrophoresis (CGE)
			3.1.3 UV Spectroscopy
		3.2 PDZ Mass Integrity by MALDI-TOF
			3.2.1 Total Mass Measurement
			3.2.2 ISD and T3 Sequencing
		3.3 Homogeneity
			3.3.1 Dynamic Light Scattering (DLS)
			3.3.2 Size Exclusion Chromatography (SEC) with Multiangle Light Scattering (SEC-MALS)
		3.4 Conformational Stability/Folding State
			3.4.1 Circular Dichroism (CD)
			3.4.2 Nano Differential Scanning Fluorimetry (nanoDSF)
			3.4.3 Differential Scanning Calorimetry (DSC)
			3.4.4 NMR
	4 Notes
	References
Chapter 7: Crystallographic Studies of PDZ Domain-Peptide Interactions of the Scribble Polarity Module
	1 Introduction
	2 Materials
		2.1 Sample Preparation for CD Analysis
		2.2 Sample Preparation for Isothermal Titration Calorimetry (ITC)
		2.3 Sample Preparation for X-Ray Crystallography
	3 Methods
		3.1 Sample Preparation for CD Spectroscopy
		3.2 Interactions of Polarity Protein PDZ Domains with Ligands
		3.3 Preparation of Cell-Polarity Protein-Peptide Complexes for Crystallization
		3.4 Crystallization of Tandem Scribble PDZ Domains Bound to Interacting Peptides
	4 Notes
	References
Chapter 8: A Fluorescence-Based Assay to Determine PDZ-Ligand Binding Thermodynamics
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Constructs, Medium, and Reagents for PDZ Domain Purification
		2.3 Reagents and Solutions
	3 Methods
		3.1 Purified CASK and Scribble PDZ Domains
		3.2 Experimental Sample Preparation
			3.2.1 Cuvette and PBM Peptide Preparation
			3.2.2 Preparation of PDZ Domain Dilution Stock Solutions
		3.3 Binding Assay Parameters and Data Collection
		3.4 Data Processing
		3.5 Data Analysis and Binding Curve Presentation
	4 Notes
	References
Chapter 9: Unveiling the Folding Mechanism of PDZ Domains
	1 Introduction
	2 Materials
		2.1 Site-Directed Mutagenesis
		2.2 Equilibrium Unfolding Experiments
		2.3 Folding Kinetic Experiments
	3 Methods
		3.1 Equilibrium Denaturation Experiments Induced by Chaotropic Agents
		3.2 Kinetic Studies
	4 Notes
	References
Chapter 10: Development of Peptide-Based PDZ Domain Inhibitors
	1 Introduction
	2 Materials
		2.1 Peptide Synthesis
		2.2 Fluorescence Polarization
		2.3 Isothermal Calorimetry
		2.4 Pull-Down of Nonischemic Brain Lysates
		2.5 Human Blood Plasma Stability Assay
		2.6 In Vitro Toxicity Experiment (MTT Assay)
		2.7 Equipment
	3 Methods
		3.1 Solid-Phase Peptide Synthesis
			3.1.1 Synthesis of PDZ Peptide Binders
			3.1.2 Generation of Peptide Probes: Conjugation with Cy5 Maleimide
		3.2 Dimeric PDZ Peptides and Cell Penetrating Tags
			3.2.1 Synthesis of Dimeric Symmetrical PDZ Binders
			3.2.2 Synthesis of the Dimeric Peptide Linker, Ns-NPEG4 Linker [10-((2-Nitrophenyl)Sulfonyl)-4,7,13,16-Tetraoxa-10-Azanonadeca...
			3.2.3 Synthesis of TAT-N-Dimeric Peptide
		3.3 Affinity and Selectivity Experiments
			3.3.1 Fluorescence Polarization Assays
			3.3.2 Isothermal Calorimetry (ITC)
		3.4 Further Validation Experiments
			3.4.1 Pull-Down of Nonischemic Brain Lysates
			3.4.2 Blood Plasma stability Assay
			3.4.3 In Vitro Toxicity Measurement (MTT-Assay)
	4 Notes
	References
Chapter 11: Dynamic Control of Signaling by Phosphorylation of PDZ Binding Motifs
	1 Introduction
	2 Materials
		2.1 In Vitro Phosphorylation of RSK1683-735
		2.2 Fluorescence Polarization (FP)
		2.3 Isothermal Titration Calorimetry (ITC)
		2.4 Protein Fragment Complementation Assay (NanoBiT)
	3 Methods
		3.1 In Vitro Phosphorylation of RSK1683-735
		3.2 Fluorescence Polarization (FP) to Study the Effects of Phosphorylation
			3.2.1 Direct Assay
			3.2.2 Competitive Assay
		3.3 Isothermal Titration Calorimetry (ITC) to Study the Effects of Phosphorylation
		3.4 NanoBiT Protein-Protein Interaction Assay to Study the Effects of Phosphorylation
	4 Notes
	References
Chapter 12: Chemical Synthesis of PDZ Domains
	1 Introduction
	2 Materials
		2.1 Plasmid Construction
		2.2 Protein Expression
		2.3 Protein Purification
		2.4 Thioester Generation
		2.5 Factor Xa Cleavage
		2.6 Solid-Phase Peptide Synthesis
		2.7 Peptide Purification
		2.8 Expressed Protein Ligation
		2.9 Desulfurization
		2.10 Equipment
	3 Methods
		3.1 Plasmid Construction for Expression of Recombinant Fragments
			3.1.1 Cloning of Plasmid Encoding C-Terminal Fragment with N-Terminal  Cys
			3.1.2 Gene Insertion into an Intein-Encoding Plasmid for the Generation of Protein Thioesters
		3.2 Protein Expression
		3.3 Protein Purification from Bacterial Lysates
			3.3.1 Cell Lysis
			3.3.2 His-Trap Purification
			3.3.3 FPLC Purification
		3.4 Protein Thioester DeltaNPDZ Formation and Isolation
		3.5 Factor Xa Cleavage to Generate N-Terminal Cys Protein Fragment DeltaCPDZ
		3.6 Solid-Phase Peptide Synthesis
		3.7 Fmoc/t-Bu-SPPS for the Synthesis of Phosphopeptides
			3.7.1 Resin Loading
			3.7.2 Fmoc-SPPS Coupling Cycle
			3.7.3 Cleavage and Global Deprotection
		3.8 Boc/Bzl-SPPS for the Insertion of Amide-to Ester Mutations
			3.8.1 Synthesis of α-Hydroxy Acids
			3.8.2 Boc-SPPS Coupling Cycle
			3.8.3 Cleavage and Global Deprotection
		3.9 Expressed Protein Ligation
			3.9.1 Oxidation of Hydrazide Peptide to Generate an Active Thioester
			3.9.2 Ligation
		3.10 Desulfurization
	4 Notes
	References
Chapter 13: Viral PDZ Binding Motifs Influence Cell Behavior Through the Interaction with Cellular Proteins Containing PDZ Dom...
	1 Introduction
	2 Cellular Processes Targeted by Viral PBMs
		2.1 Cell-Cell Junctions
		2.2 Cell Polarity
		2.3 Cell Survival and Apoptosis
		2.4 Disruption of the Immune System
	3 Relevance of CoVs Proteins Including a PBM
	4 Concluding Remarks
	References
Chapter 14: Computational Design of PDZ-Peptide Binding
	1 Introduction
		1.1 General Issues
		1.2 PDZ-Peptide Issues
		1.3 Chapter Overview
	2 High-Throughput Design of PDZ-Peptide Binding
		2.1 Model Ingredients and System Setup
			2.1.1 Energy Model
			2.1.2 Structures
			2.1.3 Unfolded Protein State
			2.1.4 Energy Matrix
			2.1.5 Monte Carlo Simulations to Explore Sequences and Structures
			2.1.6 Proteus Software Files and Documentation
		2.2 Adaptive Landscape Flattening to Design PDZ-Peptide Binding Affinity
			2.2.1 General Method
			2.2.2 Stage 1: Flattening the Unbound State
			2.2.3 Stage 2: Simulating the Bound State
			2.2.4 Application to the Tiam1-Sdc1 Complex
	3 A Medium-Throughput Design Approach
		3.1 Explicit-Solvent MD to Characterize PDZ-Peptide Complexes
			3.1.1 Conformational Restriction of the Peptide N-Terminus
			3.1.2 Force Field and MD Simulations
		3.2 Relative Binding Free Energies
			3.2.1 The Free Energy Function
			3.2.2 Fitting to Experimental Binding Free Energies
		3.3 Selected Results
			3.3.1 Mean Errors
			3.3.2 Scoring Sequences from  CPD
		3.4 Other Variants of the Model
			3.4.1 GB Instead of  PB
			3.4.2 Lazaridis-Karplus Instead of  SA
			3.4.3 Two-Trajectory Model for Peptide Flexibility
			3.4.4 Three-Trajectory Model
			3.4.5 Comparison to some PBSA or GBSA Approaches Applied to Other Systems
	4 Concluding Notes
	References
Chapter 15: Mechanoregulation of PDZ Proteins, An Emerging Function
	1 A Brief Introduction on Mechanotransduction/Mechanoregulation in Biology
	2 PDZ Proteins as Mechanotransducers
	3 Force Regulated PDZ Proteins
		3.1 ZO1
		3.2 MUPP1
		3.3 PAR3
	4 PDZ Proteins ``Second Line´´ Mechanotransducers
		4.1 PAR6
		4.2 DLG
		4.3 Afadin
		4.4 SCRIBBLE
	5 Concluding Remarks
	References
Chapter 16: Rational Design of PDZ Domain Inhibitors: Discovery of Small Organic Compounds Targeting PDZ Domains
	1 Introduction
	2 PDZ Domains as Potential Drug Targets
	3 Conclusion
	References
Index