INTRINSICALLY DISORDERED PROTEINS : methods and protocols.

Preface
Acknowledgements
Contents
Contributors
Part I: Sequence Properties
	Chapter 1: Disorder for Dummies: Functional Mutagenesis of Transient Helical Segments in Disordered Proteins
		1 Introduction
		2 Materials
		3 Methods
			3.1 Disorder Predictions and Sequence Alignments
			3.2 Prediction of Transient Helicity
			3.3 Measuring Transient Helicity with CD Spectroscopy
			3.4 Measuring Transient Helicity with NMR
			3.5 Mutant Design
		4 Notes
		References
	Chapter 2: Computational Prediction of Intrinsic Disorder in Protein Sequences with the disCoP Meta-predictor
		1 Introduction
		2 Materials
		3 Methods
			3.1 Running disCoP
			3.2 Results Generated by disCoP
		4 Case Study
		5 Notes
		References
	Chapter 3: Computational Prediction of Disordered Protein Motifs Using SLiMSuite
		1 Introduction
		2 Materials
			2.1 SLiMSuite Installation
			2.2 SLiMSuite Directory Structure
			2.3 Python 2.x
			2.4 SLiMSuite Servers
			2.5 SLiMSuite Tools
				2.5.1 SLiMProb: Prediction of Predefined SLiMs
				2.5.2 SLiMFinder: De Novo SLiM Prediction
				2.5.3 QSLiMFinder: Query-Focused De Novo SLiM Prediction
				2.5.4 CompariMotif: Motif-Motif Comparisons
				2.5.5 GABLAM: Global Alignment from BLAST Local Alignment Matrix
				2.5.6 GOPHER: Generation of Orthologous Proteins from Homology-Based Estimation of Relationships
				2.5.7 SLiMFarmer: SLiMSuite Job Farming Wrapper
				2.5.8 SLiMParser: SLiMSuite REST Job Generation and Parsing Tool
			2.6 SLiMSuite Dependencies
				2.6.1 BLAST+ Homology Search
				2.6.2 IUPred Disorder Predictor
				2.6.3 ClustalW2 Multiple Sequence Alignment
				2.6.4 ClustalOmega Multiple Sequence Alignment
				2.6.5 R
			2.7 Input Data for Motif Prediction
				2.7.1 Data Types
					Multi-protein Datasets
					Single Proteins
					Multiple Multi-protein Datasets
					Proteomes
					Motif Data
				2.7.2 SLiMSuite FASTA Format
				2.7.3 UniProt Text Files
				2.7.4 Motif Formats
				2.7.5 Example Data Table
		3 Methods
			3.1 Disorder Masking
				3.1.1 Masking from Disorder Prediction Scores
				3.1.2 Masking UniProt Features
				3.1.3 Custom Masking of FASTA Files
			3.2 Disordered Motif Prediction
				3.2.1 Prediction of New Disordered Instances of Known SLiMs (SLiMProb)
				3.2.2 Predicting Novel SLiMs De Novo in a Set of Proteins (SLiMFinder)
				3.2.3 Query-Focused De Novo SLiM Prediction (QSLiMFinder)
				3.2.4 Restricting De Novo SLiM Prediction to a Specific Protein Region (QSLiMFinder)
				3.2.5 Identifying Known Motifs from De Novo Predictions (CompariMotif)
				3.2.6 Comparing Different De Novo Prediction Runs (CompariMotif)
				3.2.7 Searching De Novo Predictions Against Another Set of Proteins (SLiMProb)
		4 Notes
		References
	Chapter 4: How to Annotate and Submit a Short Linear Motif to the Eukaryotic Linear Motif Resource
		1 Introduction
		2 Materials
			2.1 Material Required to Submit a Motif to ELM
		3 Methods
			3.1 How to Annotate and Submit a Motif Instance
			3.2 How to Annotate and Submit a Motif Class
			3.3 How to Submit a Candidate Motif Class
			3.4 How to Update a Motif Class
		4 Notes
		References
	Chapter 5: Analyzing the Sequences of Intrinsically Disordered Regions with CIDER and localCIDER
		1 Introduction
		2 Materials
		3 Methods
			3.1 Using the CIDER Webserver
			3.2 Using the localCIDER Software Package
			3.3 Sequence Parameters Calculated by CIDER
				3.3.1 Fraction of Charged Residues
				3.3.2 Net Charge per Residue
				3.3.3 Kappa (κ)
				3.3.4 Fraction Hydropathy and Fraction Disorder-Promoting Residues
				3.3.5 Diagram of States
				3.3.6 The localCIDER Software Package
				3.3.7 Example: A Case Study on p27100-198
				3.3.8 Limitations of CIDER Sequence Analysis
				3.3.9 Additional Approaches/Principles for the Analysis of Disordered Protein Sequences
		4 Notes
		References
	Chapter 6: Exploring Protein Intrinsic Disorder with MobiDB
		1 Introduction
		2 Materials
		3 Methods
			3.1 Discovering Protein Intrinsic Disorder Using MobiDB 3.0
				3.1.1 Curated Data
				3.1.2 Derived Data
				3.1.3 Predicted Data
				3.1.4 Consensus Data
			3.2 Applications
				3.2.1 Working Case: Beta-Catenin
				3.2.2 Disorder Flavors
				3.2.3 MobiDB-Lite Could Help to Identify Order-Disorder Transitions
		4 Notes
		References
Part II: Evolution
	Chapter 7: An Easy Protocol for Evolutionary Analysis of Intrinsically Disordered Proteins
		1 Introduction
		2 Materials
		3 Methods
			3.1 Finding Homologous Sequences
			3.2 Using BLAST to Generate a Dataset
				3.2.1 Isoform Selection
				3.2.2 Renaming Headers
			3.3 Making a High-Quality Multiple Sequence Alignment
				3.3.1 Multiple Sequence Alignment Construction
				3.3.2 Multiple Sequence Alignment Visualization
			3.4 Building a Phylogenetic Tree
				3.4.1 Phylogenetic Reconstruction
				3.4.2 Tree Visualization
				3.4.3 Tree Analysis
			3.5 Predicting Disorder
				3.5.1 Intrinsic Disorder Prediction
			3.6 Evolutionary Dynamics of Intrinsic Disorder
			3.7 Reflections and Future Directions
		4 Notes
		References
Part III: Production
	Chapter 8: Expression and Purification of an Intrinsically Disordered Protein
		1 Introduction
		2 Materials
			2.1 Bacterial Host and Plasmid Construct
			2.2 Rich Bacterial Media
			2.3 M9 Minimal Media
			2.4 Purification Steps 1 and 2
			2.5 High-Performance Liquid Chromatography (HPLC) Buffers
			2.6 Ulp1 Purification
			2.7 Tris-Tricine Acrylamide Gel
		3 Methods
			3.1 Protein Expression
			3.2 Cell Lysis
			3.3 Purification Step 1
			3.4 Purification Step 2
			3.5 Ulp1 Expression and Purification
			3.6 Making a Tris-Tricine Polyacrylamide Gel
			3.7 Tris-Tricine Polyacrylamide Gel Electrophoresis
		4 Notes
		References
	Chapter 9: Production of Intrinsically Disordered Proteins for Biophysical Studies: Tips and Tricks
		1 Introduction
			1.1 Considerations to Keep in Mind Before Going to the Laboratory
				1.1.1 Length of Primary Structure
				1.1.2 Amino Acid Composition
				1.1.3 Preventing Unwanted Degradation
		2 Materials
			2.1 Denaturing-Based Approaches
				2.1.1 Heat Treatment
				2.1.2 Isoelectric Precipitation
			2.2 Chromatography-Based Approaches
				2.2.1 Ion-Exchange Chromatography
				2.2.2 Reversed-Phase Chromatography
		3 Methods
			3.1 Denaturing-Based Approaches
				3.1.1 Purification by Heat Treatment
				3.1.2 Isoelectric Precipitation
			3.2 Chromatography-Based Approaches
				3.2.1 Ion-Exchange Chromatography
				3.2.2 Reversed-Phase Chromatography
		4 Notes
		References
	Chapter 10: Recombinant Production of Monomeric Isotope-Enriched Aggregation-Prone Peptides: Polyglutamine Tracts and Beyond
		1 Introduction
		2 Materials
			2.1 Transformation and Expression
			2.2 Purification and Monomerization
			2.3 NMR Sample Preparation
		3 Methods
			3.1 Transformation of E. coli Competent Cells
			3.2 Expression of the His6-Sumo-Qn Fusion Construct in Isotope-Enriched Minimal Medium
			3.3 Purification of the Isolated Qn Peptides
			3.4 Purity Polishing and Monomerization of the Qn Peptides
			3.5 Sample Preparation for NMR Measurements
		4 Notes
		References
	Chapter 11: Cell-Free Protein Synthesis of Small Intrinsically Disordered Proteins for NMR Spectroscopy
		1 Introduction
		2 Materials
			2.1 General Considerations and Necessary Material and Equipment
			2.2 Extract Cultivation
			2.3 Extract Preparation and Mg2+ Optimum Determination
			2.4 CFPS Reagents
		3 Methods
			3.1 Extract Cultivation
			3.2 Extract Preparation
			3.3 Plasmid Preparation
			3.4 CFPS Reaction Setup
			3.5 Extract Activity Test and Mg2+ Optimization
			3.6 Expression Assessment
		4 Notes
		References
Part IV: Dynamics, Ensembles, and Structures
	Chapter 12: Structural Analyses of Intrinsically Disordered Proteins by Small-Angle X-Ray Scattering
		1 Introduction
		2 Materials
			2.1 Batch Mode
			2.2 SEC-SAXS Mode
		3 Methods
			3.1 SAXS Measurements in Batch Mode
			3.2 Size-Exclusion Chromatography Coupled to SAXS (SEC-SAXS)
			3.3 Primary Data Analysis
				3.3.1 Checking for Concentration-Dependent Effects
				3.3.2 Initial Estimates of Flexibility
				3.3.3 Pair-Wise Distance Distribution Function
			3.4 Ensemble Optimization Method for the Analysis of SAXS Profiles
				3.4.1 Generation of an Ensemble of Conformations
				3.4.2 Selection of a Sub-ensemble of Conformations that Describes the SAXS Data
				3.4.3 Quantitative Estimation of Flexibility of the Ensemble
		4 Notes
		References
	Chapter 13: Determining Rg of IDPs from SAXS Data
		1 Introduction
		2 Materials
		3 Methods
			3.1 General Steps to Determine Radius of Gyration
			3.2 Determination of Rg of Histatin 5 by the Guinier Approach Using PRIMUS
			3.3 Determination of the Pair Distance Distribution Function in PRIMUS
		4 Notes
		References
	Chapter 14: Obtaining Hydrodynamic Radii of Intrinsically Disordered Protein Ensembles by Pulsed Field Gradient NMR Measuremen...
		1 Introduction
		2 Materials
			2.1 Instruments
			2.2 Materials and Solutions
			2.3 Software
		3 Methods
			3.1 Gradient Calibration
			3.2 Sample Preparation
				3.2.1 Lyophilization
				3.2.2 Sample Dilution
			3.3 Diffusion Measurement and Hydrodynamic Radius Determination
				3.3.1 Absolute Diffusion Constant
				3.3.2 Internal Standard
		4 Notes
		References
	Chapter 15: Quantitative Protein Disorder Assessment Using NMR Chemical Shifts
		1 Introduction
		2 Materials
			2.1 Computation of RCCS Reference Values Using the POTENCI Web Application
			2.2 Computation of RCCS Reference Values Using a Command-Line Python Script
			2.3 Computation of CheZOD Z-Scores Using a Web Application
			2.4 Computation of CheZOD Z-Scores from a Command-Line Interface Python Script
		3 Methods
			3.1 Computation of RCCS Reference Values Using a Web Application
			3.2 Computation of RCCS Reference Values Using a Command-Line Python Script
			3.3 Computation of CheZOD Z-Scores Using a Web Application
			3.4 Computation of CheZOD Z-Scores from a Command-Line Interface Python Script
			3.5 Interpretation of the CheZOD Z-Scores
		4 Notes
		References
	Chapter 16: Determination of pKa Values in Intrinsically Disordered Proteins
		1 Introduction
		2 Materials
			2.1 Media and Reagents
			2.2 Buffers
			2.3 Other Materials
		3 Methods
			3.1 Protein Expression in M9 Minimal Media
			3.2 Cell Lysis and Protein Purification
			3.3 NMR Spectroscopy
				3.3.1 Sample Preparation
				3.3.2 1H-15N Heteronuclear Single Quantum Correlation (HSQC) Spectra for Protein Backbone Assignment
				3.3.3 Aromatic 1H-13C HSQC Spectra for Measuring his pKas
				3.3.4 2D 13COi/1HNi+1 Spectra for Measuring Asp and Glu pKas
			3.4 Data Processing, Analysis, and Model Fitting
				3.4.1 Processing and Annotating NMR Spectra
				3.4.2 Model Fitting and pKa Determination Using MATLAB
		4 Notes
		References
	Chapter 17: Paris-DÉCOR: A Protocol for the Determination of Fast Protein Backbone Amide Hydrogen Exchange Rates
		1 Introduction
		2 Materials
		3 Methods
			3.1 Data Acquisition
			3.2 Data Processing
			3.3 Determination of Hydrogen Exchange Rates by Numerical Fitting
		4 Notes
		References
Part V: Ensembles by Computation
	Chapter 18: Predicting Conformational Properties of Intrinsically Disordered Proteins from Sequence
		1 Introduction
			1.1 R1: FCR < 0.25 and |NCPR| < 0.25; Globules and Tadpoles
			1.2 R2: 0.25  FCR  0.35 and |NCPR|  0.35; Range from Globules to Coils Depending on Context
			1.3 R3: FCR > 0.35 and |NCPR|  0.35; Charge Patterning Dictates Compaction from Coil to Hairpin
			1.4 R4: FCR > 0.35 and |NCPR| > 0.35; Coils and Semiflexible Rods
		2 Materials
			2.1 IDR Sequence(s)
			2.2 Sequence Analysis Tools
		3 Methods
			3.1 Predicting Qualitative Conformational Properties from Global Amino Acid Composition
			3.2 Classifying Experimentally Determined IDRs within the Diagram of States
			3.3 Extracting Conformational Information from Charge Patterning
				3.3.1 Calculating κ
				3.3.2 Calculating SCD
				3.3.3 Comparing κ and SCD
				3.3.4 How Do κ and SCD Correlate with Conformational Properties?
				3.3.5 Weaknesses of κ
				3.3.6 Weaknesses of SCD
			3.4 Extracting Conformational Information from Other Sequence Patterns
				3.4.1 Patterning of Expansion Driving Residues
				3.4.2 General Patterning
			3.5 Applying Patterning Metrics to Understand the Effects of Phosphorylation on Conformation
			3.6 Extracting Quantitative Conformational Information from Sequence
				3.6.1 Extracting Rh from Sequence
				3.6.2 Comparing Conformation to a Reference Coil: Chain Expansion Parameter
			3.7 The Future of Predicting Conformation from Sequence
		4 Notes
		References
	Chapter 19: Enhanced Molecular Dynamics Simulations of Intrinsically Disordered Proteins
		1 Introduction
		2 Materials
			2.1 Software
		3 Methods
			3.1 Preparation of the Molecular System
			3.2 Simulations
		4 Notes
		References
	Chapter 20: Computational Protocol for Determining Conformational Ensembles of Intrinsically Disordered Proteins
		1 Introduction
		2 Materials
			2.1 Force Field
			2.2 Simulation Software
			2.3 Experimental Data
		3 Methods
			3.1 Ensemble Reweighting
				3.1.1 Generation of Initial Ensemble
				3.1.2 Reweighting Scheme
				3.1.3 Avoiding Overfitting
				3.1.4 How to Actually Find the Optimal Weights?
				3.1.5 Assessing the Final Weights
			3.2 Validating and Using the Reweighted Ensemble
		4 Notes
		References
	Chapter 21: Computing, Analyzing, and Comparing the Radius of Gyration and Hydrodynamic Radius in Conformational Ensembles of ...
		1 Introduction
		2 Materials
			2.1 Experimental Data and Sequence of Sic1
			2.2 Software
		3 Methods
			3.1 Generating Ensembles
			3.2 Calculating Rg and Rh from Ensembles
			3.3 A Bayesian/Maximum Entropy Approach
			3.4 Calculating SAXS Data from Ensembles
			3.5 Reweighting Sic1 Ensembles Against SAXS and NMR Diffusion Experiments
			3.6 Summary
		4 Notes
		References
Part VI: Determinants of Interactions
	Chapter 22: Binding Thermodynamics to Intrinsically Disordered Protein Domains
		1 Introduction
			1.1 Isothermal Titration Calorimetry
			1.2 Thermodynamic Information Obtained from ITC at One Temperature
			1.3 ITC Results for gp120
			1.4 Determination of the Change in Heat Capacity Associated with Binding
			1.5 Experimental Design
		2 Materials
			2.1 Reagents and Supplies
			2.2 Isothermal Titration Calorimeter
			2.3 Experimental Systems
		3 Methods
			3.1 Sample Preparation
			3.2 Experimental Procedure
			3.3 Analysis of the Data
			3.4 Estimation of the Degree of Conformational Structuring
		4 Notes
		References
	Chapter 23: Analysis of Multivalent IDP Interactions: Stoichiometry, Affinity, and Local Concentration Effect Measurements
		1 Introduction
		2 Materials
			2.1 Buffers
			2.2 Proteins
			2.3 Equipment
			2.4 Software
		3 Methods
			3.1 Protein Production
			3.2 NMR Titration for Affinity Measurement
			3.3 ITC Titration for Affinity measument
			3.4 Determination of Stoichiometry
			3.5 Estimation of Local Concentration Effect
			3.6 Combining Results from the Different Analyses
		4 Notes
		References
	Chapter 24: NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions
		1 Introduction
		2 Materials
			2.1 NMR Spectrometer
			2.2 NMR Tubes
			2.3 NMR Samples
			2.4 Software
		3 Data Acquisition
			3.1 Sample Concentrations
			3.2 Experimental Setup
			3.3 Performing the NMR Titration
			3.4 Data Analysis
				3.4.1 Analysis of 1D 1H Spectra
				3.4.2 Processing
				3.4.3 Two-Dimensional Lineshape Analysis
		4 Notes
		References
	Chapter 25: Measuring Effective Concentrations Enforced by Intrinsically Disordered Linkers
		1 Introduction
		2 Materials
			2.1 DNA Constructs
			2.2 Expression and Purification of Biosensor
			2.3 Expression and Purification of Competitor Peptide
			2.4 Measurement of Effective Concentration
		3 Methods
			3.1 Cloning of Linker Sequence into Reporter Plasmid
			3.2 Expression and Purification of Fluorescent Biosensor
				3.2.1 Biosensor Expression
				3.2.2 Generation of Lysate
				3.2.3 Biosensor Purification by Ni-Affinity Chromatography
				3.2.4 Biosensor Purification by Strep-Tag Affinity Chromatography
			3.3 Expression and Purification of MBD2 Peptide
			3.4 Competition Titration
			3.5 Data Analysis
		4 Notes
		References
	Chapter 26: Determining the Protective Activity of IDPs Under Partial Dehydration and Freeze-Thaw Conditions
		1 Introduction
		2 Material
			2.1 Stock Solutions
			2.2 Treatment Buffers
			2.3 Protein Solutions
			2.4 Reaction Buffers
			2.5 Equipment
		3 Methods
			3.1 Master Mix Preparation
			3.2 Low Water Availability Assay: Freeze-Thaw Treatment
			3.3 Low Water Availability Assay: Dehydration Treatment
			3.4 Measurement of LDH or ADH Activity: Spectrophotometer Setting
			3.5 Measurement of LDH or ADH Activity: Enzyme Activity Quantification
		4 Notes
		References
	Chapter 27: Screening Intrinsically Disordered Regions for Short Linear Binding Motifs
		1 Introduction
		2 Materials
			2.1 General Materials
			2.2 Purification of du-ssDNA
			2.3 PCR Amplification of Custom-Designed Oligonucleotide Pool and dsDNA Quantification
			2.4 Synthesis of dsDNA Phagemid Library
			2.5 Electroporation and Propagation of the ProP-PD Library
			2.6 High-Throughput Expression and Purification
			2.7 ProP-PD Selections and Pooled Phage ELISA
			2.8 Preparing Samples for Next-Generation Sequencing (NGS)
		3 Methods
			3.1 Phage Library Construction
				3.1.1 Purification of du-ssDNA
				3.1.2 PCR Amplification of the Custom-Designed Oligonucleotide Pool and dsDNA Quantification
				3.1.3 Synthesis of a dsDNA Phagemid Library
				3.1.4 Electroporation and Propagation of Phage Library
			3.2 High-Throughput Expression and Purification of Bait Proteins
				3.2.1 Protein Expression
				3.2.2 Protein Purification
			3.3 HTP ProP-PD Selection and Pooled Phage ELISA
				3.3.1 Phage Display Selection
				3.3.2 Phage Pool ELISA (Day 5)
			3.4 Preparing Samples for NGS
				3.4.1 Barcoding and Amplification
				3.4.2 Normalization, Pooling, and Cleaning of Amplified Products
					Normalization of Samples
					Pooling and Cleaning
			3.5 NGS Data Handling
				3.5.1 NGS Data Demultiplexing and Cleanup
				3.5.2 Data Analysis and Storage
		4 Notes
		References
Part VII: Interactions on Surfaces
	Chapter 28: Probing IDP Interactions with Membranes by Fluorescence Spectroscopy
		1 Introduction
			1.1 Tau: An Intrinsically Disordered Protein
			1.2 Tau-Membrane Interactions
			1.3 Application of Fluorescence Spectroscopy for Probing Tau-Membrane Interactions
		2 Materials
			2.1 Recombinant Protein Production and Purification
			2.2 Small Unilamellar Vesicle (SUV) Preparation
			2.3 Acrylodan Fluorescence
			2.4 Tryptophan Fluorescence
		3 Methods
			3.1 Recombinant Protein Expression and Purification
				3.1.1 1 L Growth of Unlabeled Tau Protein
				3.1.2 Purification of Unlabeled Tau from 1 L
				3.1.3 Site-Directed Mutagenesis
			3.2 SUV Lipid Vesicle Preparation
			3.3 Acrylodan Fluorescence
			3.4 Tryptophan Fluorescence
			3.5 Data Analysis
		4 Notes
		References
	Chapter 29: Protocol for Investigating the Interactions Between Intrinsically Disordered Proteins and Membranes by Neutron Ref...
		1 Introduction
		2 Materials
		3 Methods
			3.1 NR Cell and Substrate Cleaning
			3.2 POPC Vesicle Suspension
			3.3 Formation of the Supported Lipid Bilayer
			3.4 Interaction Between NHE1-LID and the POPC Lipid Bilayer
			3.5 Data Analysis
		4 Notes
		References
	Chapter 30: Interactions of IDPs with Membranes Using Dark-State Exchange NMR Spectroscopy
		1 Introduction
			1.1 Application of NMR for Probing IDP-Membrane Interactions
			1.2 Membrane Interactions of Intrinsically Disordered Proteins
		2 Materials
			2.1 NMR
		3 Methods
			3.1 NMR
				3.1.1 1H-15N HSQC Sample Preparation
				3.1.2 NMR Spectrometer Setup
				3.1.3 1H-15N HSQC Experimental Run and Setup
				3.1.4 15N-T2 Relaxation Experimental Setup
				3.1.5 15N-DEST Experimental Setup
			3.2 Data Analysis
				3.2.1 NMR Signal Processing
				3.2.2 NMR Spectral Peak Picking
				3.2.3 NMR Data Analysis and Interpretation: 1H-15N HSQC Intensity Ratios
				3.2.4 NMR Data Analysis and Interpretation: 15N-T2 Relaxation
				3.2.5 NMR Data Analysis and Interpretation: 15N-DEST
		4 Notes
		References
Part VIII: Binding Kinetics and Mechanisms
	Chapter 31: Determination of Binding Kinetics of Intrinsically Disordered Proteins by Surface Plasmon Resonance
		1 Introduction
		2 Materials
		3 Methods
			3.1 Immobilization
			3.2 Experimental Measurements
				3.2.1 Single Cycle Protocol: Non-steady-State Kinetics
				3.2.2 Single Cycle Protocol: Affinity at Steady-State Binding
			3.3 Data Analysis
				3.3.1 Data Analysis of Non-steady-State Kinetics (Figs. 1 and 2)
				3.3.2 Data Analysis of Equilibrium Binding Constants at Steady-State Binding (Fig. 3)
		4 Notes
		References
	Chapter 32: Measuring and Analyzing Binding Kinetics of Coupled Folding and Binding Reactions Under Pseudo-First-Order Conditi...
		1 Introduction
		2 Materials
			2.1 Chemicals and Reactants
			2.2 Instruments
			2.3 Additional Equipment
		3 Method
			3.1 Equilibrium Measurements
			3.2 Instrument Setup for Stopped-Flow Mixing Experiments
			3.3 Sample Preparations for Stopped-Flow Mixing Experiments
			3.4 Association Kinetics Under Pseudo-First-Order Conditions
			3.5 Dissociation Kinetics by Displacement Under Pseudo-First-Order Conditions
		4 Notes
		References
	Chapter 33: Understanding Binding-Induced Folding by Temperature Jump
		1 Introduction
		2 Materials
		3 Methods
			3.1 Protein Samples Preparation
			3.2 Setup of the Instrument Optics
			3.3 Setup of Discharge Unit
			3.4 Temperature-Jump Experiment: Data Acquisition and Analysis
			3.5 Analysis of kobs Dependence in Pseudo-First-Order Binding Experiments
		4 Notes
		References
	Chapter 34: Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy
		1 Introduction
			1.1 Theoretical Descriptions of Chemical Exchange
			1.2 Relaxation-Compensated CPMG Dispersion Experiments for Studying Chemical Exchange
			1.3 The Carver and Richards Equation
			1.4 GLOVE
			1.5 Kinetic Models of Protein-Protein Interactions
		2 Materials
			2.1 Sample and Buffer
			2.2 Spectrometer and Software
			2.3 Pulse Sequence for the Relaxation-Compensated Constant-Time CPMG Experiment
		3 Methods
			3.1 Sample Preparation
			3.2 Identification of the Appropriate Stoichiometric Range for the CPMG Titration Experiment
				3.2.1 The HSQC Spectrum of the IDP Is Well-Resolved in Both the Free and Bound Form
				3.2.2 The HSQC Spectrum of the IDP Is Well-Resolved in Both the Free and Bound Form
				3.2.3 The HSQC Spectrum of the IDP Is Well-Resolved in the Free Form, but Severe Line-Broadening Is Observed in the Bound Form
			3.3 CPMG Titration Data Acquisition
			3.4 Dispersion Data Fitting Using the GLOVE Software
				3.4.1 Initial Data Processing
				3.4.2 Extract Peak Intensities Using pkfit or fudaFIT
				3.4.3 Convert Peak Intensity to  Using cpmg2glove
				3.4.4 Fitting of Titration Points at Each Concentration Ratio
				3.4.5 Fitting of a Titration Series to the Binding Model of Choice
		4 Notes
		References
Part IX: Higher Order-Phase Separation and Fibrillation
	Chapter 35: Determination of Protein Phase Diagrams by Centrifugation
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Stock Solutions
			2.3 Working Solutions
		3 Methods
			3.1 Equipment Setup
			3.2 Sample Preparation
			3.3 Data Collection
				3.3.1 Collection of Light Phase Concentration Data
				3.3.2 Collection of Dense Phase Concentration Data
			3.4 Data Processing
			3.5 Example Calculations
				3.5.1 Sample Preparation
					First-Time Mapping of a Phase Diagram
					Subsequent Data Collections
				3.5.2 Calculating Concentration from A280 Readings
				3.5.3 Calculating Error Bars
		4 Notes
		References
	Chapter 36: In Vitro Transition Temperature Measurement of Phase-Separating Proteins by Microscopy
		1 Introduction
		2 Materials
			2.1 Proteins and Buffers
			2.2 Equipment
			2.3 Software
		3 Methods
			3.1 Microscope and Thermal Stage Setup
			3.2 Sample Preparation
			3.3 Data Collection
			3.4 Analysis
		4 Notes
		References
	Chapter 37: Walking Along a Protein Phase Diagram to Determine Coexistence Points by Static Light Scattering
		1 Introduction
		2 Materials
			2.1 Protein Sample Preparation
			2.2 Static Light Scattering Measurements
			2.3 Absorbance Measurements at 280 nm
		3 Methods
			3.1 Buffer Exchange for Protein That Is Under Denaturing Conditions
			3.2 Preparation of the Light Scattering Instrument
			3.3 Sample Preparation for Light Scattering
			3.4 Light Scattering Measurements
			3.5 Data Analysis
		4 Notes
		References
	Chapter 38: Expression and Purification of Intrinsically Disordered Aβ Peptide and Setup of Reproducible Aggregation Kinetics ...
		1 Introduction
		2 Materials
			2.1 Luria Broth (LB) Medium
			2.2 LB/Agar Plates
			2.3 Overnight Express Medium for Auto Induction
			2.4 M9 Minimal Medium
			2.5 Chromatography Resins, Columns, Chemicals, and Equipment
			2.6 Buffers
		3 Methods
			3.1 Ca2+-Competent E. coli Cells
			3.2 Transformation of the Expression Plasmid into E. coli
			3.3 Expression in Overnight Express Medium for Auto Induction (See Note 20)
			3.4 Expression of Aβ(M1-42) Peptide in M9 Minimal Medium (See Note 20)
			3.5 Expression of Aβ Peptide in LB Medium (See Note 33)
			3.6 Purification of Aβ(M1-40) or Aβ(M1-42) Without Urea (See Notes 34 and 35)
			3.7 Purification of Aβ(M1-40) or Aβ(M1-42) with Urea
			3.8 Modified Protocol for More Aggregation-Prone Aβ Variants (A2V, E22G, etc.)
			3.9 Modified Protocol for Aβ Variants with a Different Net Charge (D7N, E22G, E22K, etc.)
			3.10 Preparation of Samples for Kinetics Experiments
			3.11 Kinetic Experiment by ThT Fluorescence
			3.12 Kinetics Experiments with Pre-formed Seeds
			3.13 Outsourced Synthesis and Cloning of Gene for Amyloid β Peptide Expression
		4 Notes
		References
	Chapter 39: Measuring Interactions Between Tau and Aggregation Inducers with Single-Molecule Förster Resonance Energy Transfer
		1 Introduction
		2 Materials
			2.1 Materials for Sample Preparation and Purification
				2.1.1 Plasmids and Strains
				2.1.2 Solutions, Media, and Buffers
			2.2 SmFRET Instrument
		3 Methods
			3.1 PEG-PLL
			3.2 smFRET Standards
			3.3 Design of Tau Constructs for smFRET Measurements
				3.3.1 Introduction of Cysteines for Labeling
				3.3.2 Creation of Tau Isoforms and Fragments
			3.4 Tau Expression and Purification
			3.5 Labeling Tau for smFRET Measurements
			3.6 Preparation of Coverslips
			3.7 Checking Alignment by Measuring Diffusion Times of Fluorescent Standards
			3.8 Calibrate Instrument with smFRET Standards
			3.9 SmFRET Measurements of Tau
			3.10 Analysis of smFRET Data
			3.11 SmFRET Measurements with polyP or Other Aggregation Inducers
		4 Notes
		References
Part X: Modification and Targeting
	Chapter 40: Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Phos-tag SDS-PAGE
		1 Introduction
		2 Materials
			2.1 Mn2+-Phos-tagTM SDS-PAGE gels
			2.2 Zn2+-Phos-tagTM SDS-PAGE Gels
			2.3 Colloidal Coomassie Staining
			2.4 In Vitro Kinase Assay and Autoradiography
		3 Methods
			3.1 In Vitro Kinase Assay
			3.2 Preparing Phos-tagTM SDS-PAGE Gels
			3.3 Electrophoresis
			3.4 Post-electrophoresis Methods
				3.4.1 Coomassie Staining
				3.4.2 Western Blotting
				3.4.3 Autoradiography
		4 Notes
		References
	Chapter 41: Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR
		1 Introduction
		2 Materials
			2.1 Stock Solutions Preparation and Storage
			2.2 Sample Preparation
			2.3 NMR Spectra Acquisition and Analysis
		3 Methods
			3.1 Preparation of the IDP Stock
			3.2 Optimization of Phosphorylation Conditions
			3.3 Assignment of NMR Spectra in the Conditions Used for Phosphorylation Reactions
			3.4 Phosphorylation Monitoring by NMR
				3.4.1 Phosphorylation Monitoring Using Continuous NMR Readout
				3.4.2 Phosphorylation Monitoring Using Quenched Reactions
			3.5 Analysis of NMR Data
		4 Notes
		References
	Chapter 42: Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Quantitative Mass-Spectrometry
		1 Introduction
		2 Materials
			2.1 In Vitro Kinase Assay
			2.2 SDS Polyacrylamide Gels
			2.3 Coomassie Blue G-250 Staining
			2.4 In-Gel Digestion
			2.5 Sample Clean-Up on StageTips
			2.6 LC-MS/MS Analysis
		3 Methods
			3.1 Kinase Assay
			3.2 SDS Polyacrylamide Gel Electrophoresis
			3.3 Coomassie Blue G-250 Staining
			3.4 Autoradiography
			3.5 In-Gel Digestion of Proteins
			3.6 Sample Clean-Up on StageTips
			3.7 LC-MS/MS Analyses
			3.8 Mass Spectrometry Data Analysis
		4 Notes
		References
	Chapter 43: Targeting an Intrinsically Disordered Protein by Covalent Modification
		1 Introduction
		2 Materials
			2.1 Warheads
			2.2 Covalent Modification of Calpastatin and Calpain Inhibition
		3 Methods
			3.1 Characterization of Warheads
			3.2 Covalent Modification of Calpastatin
				3.2.1 Modification Reaction
				3.2.2 Following Modification by DTNB
				3.2.3 Following Protein Modification by MS
					Mass Determination of Proteins
					Protein Sequence Analysis
				3.2.4 Identification of the Modification by SDS-PAGE
			3.3 CD Spectroscopy
			3.4 Inhibition Assay
				3.4.1 Determination of Michaelis-Menten Constant (Km)
				3.4.2 Inhibition Assay: Determination of Inhibitory Constant (Ki)
				3.4.3 Data Analysis
			3.5 Conclusions and Recommendations
		4 Notes
		References
Part XI: In Cell and Interactomes
	Chapter 44: Recording In-Cell NMR-Spectra in Living Mammalian Cells
		1 Introduction
		2 Materials
			2.1 Solutions and Media
			2.2 Instruments/Equipment
		3 Methods
			3.1 Expression and Purification of Labeled Proteins in Escherichia coli
			3.2 Preparation of HEK-293 Cells for Electroporation
			3.3 In-Cell NMR-Sample Preparation
			3.4 NMR Measurements
		4 Notes
		References
	Chapter 45: In-Cell NMR of Intrinsically Disordered Proteins in Mammalian Cells
		1 Introduction
		2 Materials
			2.1 Expression and Purification of 15N-Labelled N-Terminally Acetylated αSyn in Bacteria
				2.1.1 Equipment and Reagents for Transformation
				2.1.2 Equipment and Reagents for Preculture
				2.1.3 Equipment and Reagents for LB Culture
				2.1.4 Equipment and Reagents for Minimal Media-Culture and Induction
				2.1.5 Equipment and Reagents for Rapid Purification of αSyn
			2.2 Cell Culture and Manipulation
				2.2.1 Equipment and Reagents for Culturing HEK-293 Cells
				2.2.2 Equipment and Reagents for αSyn Electroporation
				2.2.3 Equipment and Reagents for Cell Harvesting and Collection into the NMR Tube
				2.2.4 Equipment and Reagents for NMR
		3 Methods
			3.1 αSyn Expression and Purification
				3.1.1 Bacterial Transformation
				3.1.2 Preculture, 2x LB culture, and Expression in Minimal Media
				3.1.3 Periplasmic Purification
				3.1.4 Protein Clearance by Heat Shock
				3.1.5 Protein Clearance by Precipitation with AS
			3.2 αSyn Intracellular Delivery (See Note 12)
				3.2.1 Cell Culture
				3.2.2 αSyn Delivery by Electroporation
			3.3 Cell Collection and NMR
				3.3.1 Cell Collection
				3.3.2 Setting Up the Spectrometer and NMR Measurements (See Note 23)
				3.3.3 Spectrum Visualization Basic Data Analysis
				3.3.4 Determination of Cell Leakage
		4 Notes
		References
	46: Analyzing IDPs in Interactomes
		1 Introduction
		2 Materials
		3 Methods
			3.1 Retrieving Sequence Information from the UniProt Database
			3.2 Generating PPIs for a Query Protein
				3.2.1 Using UniProt to Retrieve a Set of PPIs for a Query Protein
				3.2.2 Using BioGRID to Retrieve a PPI Network for a Query Protein
				3.2.3 Using IntAct to Retrieve a PPI Network for a Query Protein
				3.2.4 Using DIP to Retrieve a PPI Network for a Query Protein
				3.2.5 Using MINT to Retrieve a PPI Network for a Query Protein
				3.2.6 Using HPRD to Retrieve a PPI Network for a Query Protein
				3.2.7 Using KEGG to Search for PPI Networks (or Pathways) Associated with a Query Protein
				3.2.8 Using STRING to Search for PPI Network of a Query Protein
				3.2.9 Using APID to Search for PPI Network of a Query Protein
				3.2.10 Advantages and Limitations of the Different Network Tools
			3.3 Generating PPIs for a Set of Query Proteins
				3.3.1 Using STRING to Generate a PPI Network for a Set of Query Proteins
				3.3.2 Using APID to Generate a PPI Network for a Set of Query Proteins
			3.4 Analyzing Intrinsic Disorder Predisposition of a Query Protein (or a Set of Query Proteins) and Interactors from the Corre...
				3.4.1 Looking at the Global Disorder Status of All the Members of a PPI Network Protein
					Analysis of Protein Amino Acid Composition: Applying Compositional Profiler Tool to Obtain Fractional Amino Acid Composition o...
					Using CH-Plot and CDF Binary Predictors and a Combined CH-CDF Approach
					Using Per-residue Predictors
				3.4.2 Detailed Characterization of the Peculiarities of Disorder Distribution and Disorder-Based Functionality in a Set of Sel...
				3.4.3 Web-Based Means for the Visualization of Disorder Distribution in a Protein
				3.4.4 Intrinsic Disorder-Based Functional Analyses
		4 Notes
		References
Index