Ice Binding Proteins: Methods and Protocols (Methods in Molecular Biology, 2730)

✍ Scribed by Ran Drori (editor), Corey Stevens (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 233
• Category: Library

✦ Synopsis

This volume provides methods to study ice-binding proteins (IBPs), and applications involving these proteins. Chapters are divided into three parts describing identifying, isolating, and purifying ice-binding proteins, characterize both IBPs and ice-nucleating proteins (INPs), and synthesize mimics of IBPs, as well as applications involving these proteins. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Ice Binding Proteins: Methods and Protocols aims to provide both experimental and simulations protocols to help new researchers, break-the-ice, and enter this exciting field, while also supporting established researchers broaden the scope of theirinvestigations.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Isolating, Purifying and Crystallizing IBPs
Chapter 1: Identifying Ice-Binding Proteins in Nature
1 Introduction
2 Materials
2.1 Fish Blood Collection
2.2 Collection of Hemolymph
2.3 Identification of IBPs in Microbes Using an Ice Pitting Assay
2.4 Collection of Plant Apoplastic Fluid Containing IBPs
3 Methods
3.1 Collecting Blood from Fishes
3.2 Collection of Insect Hemolymph
3.3 Identification of IBP Activity in Microbial Spent Media by Ice Pitting of the Basal Plane of a Single Ice Crystal
3.4 Collecting Apoplastic Fluid by Leaf Infusion and Centrifugation
4 Notes
References
Chapter 2: Ice Shell Purification of Ice-Active Compounds
1 Introduction
2 Materials
2.1 Preparation of Solution Containing Ice-Binding Compounds
2.2 Ice Shell Preparation
2.3 Ice Shell Growth
3 Methods
3.1 Sample Preparation
3.2 Ice Shell Preparation
3.3 Ice Shell Growth
4 Notes
References
Chapter 3: Ice Isn´t the Only Crystal in Town: Structure Determination of Ice-Binding Proteins via X-Ray Crystallography
1 Introduction
1.1 The Importance of Structural Biology to Ice-Binding Protein Research
1.2 The Big Three of Structural Biology
2 Theory of X-Ray Crystallography
2.1 X-Ray Diffraction: The Closest We Have to an X-Ray Camera
2.2 Protein Crystals as Signal Amplifiers
2.3 Simple Explanations for Non-simple Math
3 Sample Preparation and Crystal Growth
3.1 Protein Samples: Crap In, Crap Out
3.2 The Black Magic of Crystal Growing
3.3 If the Worst Should Happen
4 Data Collection
4.1 Different Strokes: The Various Ways of Collecting Data
4.2 Variables Associated with Data Collection
4.3 Choose Your Weapon: Synchrotron vs. Home-Source Diffractometers
5 Structure Solution
5.1 Data Reduction
5.2 Solving the Phase Problem
5.3 Model Building and Refinement
6 You Have a StructureNow What?
References
Chapter 4: Large-Scale Purification of Natural Ice-Binding Proteins by the Falling Water Ice Purification Method
1 Introduction
2 Materials and Special Equipment
2.1 Special Equipment
2.2 Materials
3 Methods
3.1 Optimizing the Working Solution
3.2 Adjusting the Ice Machine
3.3 Running the FWIP First Purification Round
3.4 Analysis of Purification Efficiency
3.5 Second and Subsequent Purification Rounds
3.6 Cleaning and Maintaining the Icemaker
4 Notes
References
Part II: Measuring and Quantifying the Activity of IBPs
Chapter 5: The Nanoliter Osmometer: Thermal Hysteresis Measurement
1 Introduction
2 Materials and System Assembly
2.1 Materials
2.2 Assembly of the Nanoliter Osmometer
3 Methods
3.1 Copper Disk Cleaning
3.2 Glass Capillary Preparation
3.3 System Preparation
3.4 Sample Injection
3.5 Obtaining a Single Ice Crystal in the Droplet
3.6 Determine the TH
3.7 Calculate the Thermal Hysteresis Gap
3.8 Melting Hysteresis
3.9 Temperature Calibration
4 Notes
References
Chapter 6: Evaluation of Ice Recrystallization Inhibition of Ice-Binding Proteins by Monitoring Specific Ice Crystals
1 Introduction
2 Materials
2.1 AFP Solutions
2.2 Instrumental Setup
3 Methods
3.1 Sample Preparation
3.2 Observation of Ice Recrystallization Process
3.3 Image Analysis
3.4 Evaluation of Ice Recrystallization Efficiency
4 Notes
References
Chapter 7: Measurement of Ice Nucleation Activity of Biological Samples
1 Introduction
2 Materials
2.1 Sample Purification
2.2 Freezing Droplet Experiments
3 Methods
3.1 Sample Purification
3.2 Freezing Droplet Experiments
3.3 Analysis of Experiments
References
Chapter 8: Investigating the Interaction Between Ice-Binding Proteins and Ice Surfaces Using Microfluidic Devices and Cold Sta...
1 Introduction
2 Materials
2.1 Antifreeze Proteins
2.2 Microfluidic Device Fabrication
2.3 Cold Stage and Microscopy
3 Methods
3.1 Microfluidic Device Fabrication (Without Mold Synthesis) and Pre-freezing Preparation
3.2 Placement of Microfluidic Device in Cold Stage
3.3 Freezing the Sample and Obtaining a Single Ice Crystal
3.4 Solution Exchange around Single Crystals
4 Notes
References
Chapter 9: Quantification of the Ice Nucleation Activity of Ice-Binding Proteins Using a Microliter Droplet Freezing Experiment
1 Introduction
2 Materials
2.1 Instrument Construction
2.2 Materials for Performing a Measurement
3 Methods
3.1 Performing a Measurement
3.2 Background Measurements
3.2.1 Pure Water
3.2.2 Handling Blanks
4 Experiment Analysis
5 Notes
References
Chapter 10: Measurement of Ice-Binding Protein Activity in Highly Alkaline Environments
1 Introduction
2 Materials
2.1 Alkaline Solutions
2.1.1 Simple Alkaline Solutions
2.1.2 Complex Alkaline Solutions
2.2 IBP Integrity
2.2.1 Size-Exclusion Chromatography to Determine Degradation or Aggregation
2.2.2 SDS-PAGE to Determine Denaturation
2.2.3 Circular Dichroism to Determine Secondary Structure
2.2.4 IRI Activity Determination
2.3 Cement Mix Design
2.4 Cement Performance Characterization
2.4.1 Freeze-Thaw Performance of Cement Paste Cylinders
2.4.2 DSC for Ice Content Determination
3 Methods
3.1 Alkaline Solutions
3.2 IBP Integrity
3.2.1 SEC
3.2.2 SDS-PAGE
3.2.3 Circular Dichroism (CD)
3.2.4 IRI Activity Determination
3.3 Cement Mix Design
3.4 Cement Performance Characterization
3.4.1 Freeze-Thaw Performance
3.4.2 DSC for Ice Content Determination
4 Notes
References
Chapter 11: Measurement of Ice-Binding Protein Inhibition of Non-ice Crystal Growth
1 Introduction
2 Materials
3 Methods
3.1 How to Make an Experimental Cell
3.2 Methods of Preparing THF-Water
3.3 Methods of Preparing Sample Solutions
3.4 Methods of Preparing Sample Cell
3.5 Information About the Unidirectional Growth Apparatus
3.6 Experimental Procedure of Unidirectional Growth
3.7 Example Pictures of THF Hydrate Growth Interface Shift
3.8 How to Measure the Degree of Supercooling of the Growth Interface and Determine Its Error Bar
4 Notes
References
Chapter 12: Divergent Mechanisms of Ice Growth Inhibition by Antifreeze Proteins
1 Inhibition of Ice Growth by Additives
1.1 Adsorption Rates of AFPs to Ice and Crystal Morphology
1.2 Acceleration of Ice Growth by AFPs
1.3 Synergy Effect Enhances the Activity of AFPs
1.4 Model for Ice Growth Inhibition by AFPs
References
Part III: Chemical Modifications and Synthesis of IBP Mimics, MD Simulations and Evolution of IBPs
Chapter 13: Multiscale Molecular Dynamics Simulations of Ice-Binding Proteins
1 Introduction
2 Materials
3 Methods
3.1 System Preparation
3.1.1 All-Atom Simulations with Nonpolarizable Water Model
3.1.2 All-Atom Simulations with Polarizable Water Model
3.1.3 Coarse-Grained Simulations
4 Identification of Ice and Ice-Like Order
5 Notes
References
Chapter 14: Synthesis of Polymeric Mimics of Ice-Binding Proteins
1 Introduction
2 Materials
3 Methods
3.1 Synthesis, Purification, and Characterization of the Poly(vinyl acetate)-b-poly(acrylonitrile) Precursor PVAc-b-PAN
3.2 Synthesis, Purification, and Characterization of Poly(vinyl alcohol)-b-poly(acryl acid) (PVOH-b-PAA)
3.3 Size Exclusion Chromatography (SEC)
3.4 1H NMR Spectroscopy
4 Notes
References
Chapter 15: Generating Ice-Binding Protein-Polymer Bioconjugates
1 Introduction
1.1 Protein-Polymer Conjugates
1.2 Ice-Binding Protein-Polymer Conjugates
1.3 Available Chemistries for Generating Protein-Polymer Bioconjugates
2 Materials
2.1 Consumable Materials
2.2 Research Equipment
3 Methods
3.1 Conjugating IBP to Polymer
3.2 Physical Characterization of Protein-Polymer Conjugate
3.2.1 Size-Exclusion Chromatography Purification of Conjugate
3.3 Activity Analysis of Conjugate
4 Notes
References
Chapter 16: Analysis of Ice-Binding Protein Evolution
1 Introduction
2 Materials
3 Methods
3.1 Search for Putative IBPs
3.2 Phylogenies
3.3 Assessing Putative IBPs
3.4 Gene Mapping
4 Notes
References
Index