Peptide, Protein and Enzyme Design

 
 
Academic Press
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  • erschienen am 27. August 2016
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De Novo Enzyme Design, the newest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume includes the design of metal binding maquettes, insertion of non-natural cofactors, Cu metallopeptides, non-covalent interactions in peptide assemblies, peptide binding and bundling, heteronuclear metalloenzymes, florinated peptides, De Novo imaging agents, and protein-protein interaction.


  • Continues the legacy of this premier serial with quality chapters on de novo enzyme design
  • Represents the newest volume in the Methods in Enzymology series, providing premier, quality chapters authored by leaders in the field
  • Ideal reference for those interested in the study of enzyme design that looks at both structure and mechanism
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  • Front Cover
  • Peptide, Protein and Enzyme Design
  • Copyright
  • Contents
  • Contributors
  • Preface
  • References
  • Chapter One: Chemical Posttranslational Modification with Designed Rhodium(II) Catalysts
  • 1. Introduction
  • 2. Synthesis of Rhodium(II) Conjugates as Protein Modification Catalysts
  • 2.1. Discussion
  • 2.2. Materials and General Considerations
  • 2.2.1. Preparation of Metalation Buffer
  • 2.3. Protocol 1: Preparation of Rh2(OAc)3(tfa)1
  • 2.4. Protocol 2: Preparation of Rhodium(II) Conjugates in Organic Solution
  • 2.4.1. Preparation of a Metalated FKBP Inhibitor (Coughlin et al., 2014)
  • 2.5. Protocol 3: Preparation of Rhodium(II) Conjugates in Aqueous Solution
  • 2.5.1. Preparation of the Metallopeptide S2ERh
  • 3. Modification of an SH3 Domain and Gel-Based Visualization Thereof
  • 3.1. Discussion
  • 3.2. Materials and General Considerations
  • 3.2.1. Preparation of 1X Transfer Buffer
  • 3.2.2. Preparation of Protein Modification Buffer
  • 3.2.3. Preparation of MALDI-MS Matrix
  • 3.3. Protocol 4: Rhodium(II)-Catalyzed Protein Modification
  • 3.3.1. Modification of SH3 Domain of Yes Kinase in Mammalian Cell Lysate (Vohidov, Coughlin, & Ball, 2015)
  • 3.4. Protocol 5: Visualization of an Alkyne-Tagged Protein by Chemical Blotting
  • 3.4.1. Fluorescent ``Chemical Blot´´ Analysis of a Modified SH3 Domain
  • References
  • Chapter Two: Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considera ...
  • 1. Introduction
  • 2. General Binding Theory and Relevance of Kd
  • 3. General Pitfalls in Cell-Based Binding Assays
  • 3.1. Time to Equilibrium
  • 3.2. Ligand Depletion
  • 4. Measuring Binding on the Surface of Yeast
  • 4.1. Materials
  • 4.1.1. Yeast Cells
  • 4.1.2. Solutions and Media
  • 4.1.3. Proteins/Antibodies
  • 4.2. Method
  • 5. Measuring Binding on the Surface of Mammalian Cells
  • 5.1. Direct Binding
  • 5.2. Competition Binding
  • 6. Other Methods of Measuring Binding: Kinetic Exclusion Assay and Surface Plasmon Resonance
  • 6.1. Kinetic Exclusion Assay
  • 6.2. Surface Plasmon Resonance
  • 6.3. Comparison
  • 7. Summary
  • Acknowledgments
  • References
  • Chapter Three: Protein and Antibody Engineering by Phage Display
  • 1. Introduction
  • 2. Equipment
  • 3. Materials
  • 3.1. Cell Lines
  • 3.1.1. Protocol 1: Production of SS320 Cells (Modified from Tonikian, Zhang, Boone, & Sidhu, 2007)
  • 3.1.2. Protocol 2: Production of XL1-Blue Cells
  • 3.2. M13KO7 Helper Phage
  • 3.2.1. Protocol 3: Production of M13KO7 Helper Phage (Modified from Tonikian et al., 2007)
  • 3.3. Phagemid Considerations
  • 3.4. Reagents
  • 3.4.1. Media
  • 3.4.2. Buffers (Filter Sterilize)
  • 3.4.3. Solutions
  • 3.4.4. Antibiotics (Filter Sterilize)
  • 3.4.5. Other Reagents
  • 4. Phage Display and Library Design
  • 4.1. Humanization of a Murine SUDV Antibody (Chen et al., 2014)
  • 4.2. Mapping Hotspot Residues of Protein-Protein Interfaces (Frei et al., 2015
  • Stewart et al., 2013)
  • 4.2.1. Protocol 4: Cloning into HP153
  • 4.2.2. Protocol 5: Amplifying and Titering Phage
  • 4.2.3. Protocol 6: Phage ELISA
  • 5. Library Production
  • 5.1. Kunkel Mutagenesis
  • 5.1.1. Protocol 7: dU-ssDNA Synthesis (Modified from Tonikian et al., 2007)
  • 5.1.2. Protocol 8: Kunkel Mutagenesis (Modified from Kunkel et al., 1987
  • Tonikian et al., 2007)
  • 5.2. Primer Design
  • 5.3. Library Quality Control
  • 6. Library Selections and Screening
  • 6.1. Selection
  • 6.1.1. Protocol 9: Selection
  • 6.2. Screening
  • 6.2.1. Protocol 10: Monoclonal ELISA
  • 6.3. Characterization of Selected Clones
  • 6.3.1. Protocol 11: Expressing Soluble Fabs (Or Other Proteins) in E. coli
  • 6.3.2. Protocol 12: Expressing Fab as IgG in HEK239F Cells
  • 6.4. Troubleshooting
  • References
  • Chapter Four: Incorporation of Unnatural Amino Acids into Proteins Expressed in Mammalian Cells
  • 1. Introduction
  • 2. Fluorescence Assay for Straightforward Evaluation of Uaa-Incorporating Systems
  • 2.1. Materials
  • 2.2. Transfection of HEK293 Cells and Fluorescence Measurement
  • 2.3. Evaluation of Different UaaRS-tRNA Pairs
  • 3. Incorporation of Uaas into Membrane Proteins
  • 3.1. Materials
  • 3.2. Transfection of HEK293T Cells and Cross-Linking Procedure
  • 3.3. Confirming Uaa Incorporation and Cross-Linking Events
  • 4. Conclusion
  • References
  • Chapter Five: Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids
  • 1. Introduction
  • 2. The General Procedure for Enzyme Design with Unnatural Amino Acids
  • 3. Unnatural Amino Acid Design
  • 4. Synthetic Chemistry-Guided Unnatural Amino Acid Design
  • 5. Synthetic Methods for Unnatural Amino Acids
  • 5.1. Unnatural Amino Acid Toolkit
  • 5.2. Metal-Chelating Amino Acid
  • 5.3. Redox Mediators
  • 5.4. Click Chemistry Reaction Reagents
  • 6. Aminoacyl-tRNA Synthetase Screening
  • 7. Protein Design
  • 8. Protein Expression
  • 8.1. Plasmid Preparation
  • 8.2. Protein Expression
  • 9. Conclusion and Perspective
  • References
  • Chapter Six: Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Nov ...
  • 1. Introduction
  • 2. Materials
  • 3. Methods
  • 3.1. Synthesis and Purification
  • 3.2. Peptide Purification
  • 3.3. Protein Crystallization
  • 3.4. Data Collection and Processing
  • 3.5. Structure Determination and Refinements
  • Acknowledgments
  • References
  • Chapter Seven: SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Res ...
  • 1. Introduction
  • 2. Isopeptide-Mediated Enzyme Cyclization
  • 2.1. Partners for Spontaneous Isopeptide Bond Formation
  • 2.2. Cyclization with Isopeptide Bonds
  • 2.3. Selection of Enzymes for Cyclization
  • 2.4. Cloning Genes of Interest into the SpyRing Cassette
  • 2.5. Confirming Successful Cyclization
  • 3. Characterization of Enzyme Resilience After SpyRing Cyclization
  • 3.1. Industrial Relevance of Phytase
  • 3.2. Measuring Aggregation Can Provide Evidence for an Increase in Thermal Resilience
  • 3.3. Recovered Activity Is the Most Important Measurement When Testing Thermal Resilience
  • 3.4. FLuc Is an Example of a Poor Target for SpyRing Cyclization
  • 3.5. Dynamic Scanning Calorimetry Is a Useful Biophysical Tool to Study SpyRing Cyclization
  • 4. Concluding Remarks
  • Acknowledgments
  • References
  • Chapter Eight: Engineering and Application of LOV2-Based Photoswitches
  • 1. Introduction
  • 2. Engineering LOV2-Based Photoswitches
  • 2.1. Rational Engineering of a LOV2-Based Photoswitch
  • 2.1.1. Embedding Peptides
  • 2.1.2. Fusing Protein Domains
  • 2.2. Validating LOV2-Based Photoswitches
  • 2.2.1. Fluorescence Polarization Competition Assay to Measure Binding Affinities in the Lit and Dark States
  • 2.3. Improving Initial Switches by Known Mutations
  • 2.4. Library-Based Screening and Selection for Functional LOV2-Based Photoswitches
  • 2.4.1. Phage Display Panning
  • 2.5. Tuning the Activity Timescales of the LOV2-Based Photoswitch
  • 3. Using LOV2-Based Photoswitches
  • 3.1. Single-Cell Microscopy
  • 3.2. Image Analysis
  • 3.3. Functional Assays
  • 3.3.1. Yeast Transcription Using Photoswitch for Light-Induced Dimerization
  • 3.3.2. Tissue Culture Illumination Setup
  • 4. Summary and Perspectives
  • References
  • Chapter Nine: Minimalist Design of Allosterically Regulated Protein Catalysts
  • 1. Introduction
  • 2. Methods
  • 2.1. Required Tools
  • 2.2. Overall Approach
  • 2.3. Starting Points for the Design
  • 2.4. Testing the Possibility of the Substrate Associating with the Protein Chosen as a Starting Point for the Design
  • 2.5. Identification of the Positions to Mutate
  • 2.6. Establishing of the Impact of the Mutations on the Overall Fold
  • 2.7. Predicting the Feasibility of Catalysis
  • Acknowledgments
  • References
  • Chapter Ten: Combining Design and Selection to Create Novel Protein-Peptide Interactions
  • 1. Introduction
  • 2. Structure-Guided Rational Design
  • 3. Split Fluorescent Protein Assays for Identifying PPIs
  • 3.1. Screening Protein Libraries Using Split FPs
  • 3.1.1. Cloning
  • 3.1.2. Expression of Split GFP Components
  • 3.1.3. Screening PPIs in E. coli
  • 3.2. Quantifying PPIs Using Split FPs
  • 3.2.1. Expression and Quantification of Split GFP Components
  • 3.3. Selecting for PPIs Using Split FPs
  • 3.3.1. Cloning
  • 3.3.2. Selection
  • 4. Validation
  • 4.1. Cloning, Expression, and Purification of Hits
  • 4.2. Qualitative Measurements of Protein-Peptide Interactions
  • 4.3. Quantifying PPIs In Vitro
  • 4.4. Incorporating Redesign
  • 5. Future Perspective: Modular Design of PPI
  • 6. Conclusion
  • References
  • Chapter Eleven: Metal-Directed Design of Supramolecular Protein Assemblies
  • 1. Introduction
  • 2. Metal-Directed Protein Self-Assembly
  • 2.1. Choosing a Protein Building Block for MDPSA
  • 2.2. MDPSA Using Metal Chelating Motifs Composed of Natural Amino Acids
  • 2.3. MDPSA Using Synthetic Metal-Coordinating Ligands
  • 3. Metal-Templated Interface Redesign
  • 3.1. Redesign of Noncovalent Interfaces with MeTIR
  • 3.2. Disulfide Cross-Links to Enhance Scaffold Robustness
  • 3.3. In Vivo Assembly and Functional Screening of Metal-Templated Protein Assemblies
  • 4. Methods
  • 4.1. Cytochrome cb562 Variant Expression and Purification
  • 4.2. Iodoacetamide Ligand Synthesis and Protein Labeling
  • 4.3. Determining Oligomeric State by Sedimentation Velocity Experiments
  • 4.4. Measuring Protein-Protein Affinities with Sedimentation Equilibrium Experiments
  • 4.5. Crystallization of cyt cb562 Variants
  • 4.6. Periplasmic Extraction of cyt cb562 Assemblies from E. coli
  • 4.7. In Vivo Screening of cyt cb562 Assemblies with ß-Lactamase Activity
  • 5. Conclusion
  • Acknowledgments
  • References
  • Chapter Twelve: Designing Fluorinated Proteins
  • 1. Introduction
  • 1.1. Basis of Protein Stabilization by Fluorinated Amino Acid Residues
  • 1.2. Choice of Fluorinated Amino Acids
  • 1.3. Synthesis of Fluorinated Proteins
  • 1.4. Designing Fluorinated Amino Acids into Proteins
  • 1.5. Evaluating Structural and Thermodynamic Effects of Incorporating Fluorinated Side-Chains
  • 1.5.1. Thermodynamic Analysis
  • 1.5.2. Crystallographic Analysis
  • 1.6. Concluding Remarks
  • 2. Methods
  • 2.1. Measuring the Thermodynamic Stability of Fluorinated Proteins by Circular Dichroism
  • 2.2. Determining DeltaG from GuHCl-Induced Protein Unfolding
  • 2.2.1. Treatment of Baselines
  • 2.3. Determining ?H°', ?S°', and ?C°p from Heat and GuHCl-Induced Protein Unfolding
  • 2.3.1. Treatment of Baseplanes
  • 2.4. MATLAB Code for Determining ?G° Using GuHCl Unfolding
  • 2.5. MATLAB Code for Determining ?H°, ?S°, and ?C°p Using Heat and GuHCl Unfolding
  • Acknowledgments
  • References
  • Chapter Thirteen: Solid Phase Synthesis of Helically Folded Aromatic Oligoamides
  • 1. Introduction
  • 2. Materials
  • 2.1. Reagents
  • 2.2. Equipment
  • 2.3. Reagent Setup
  • 2.4. Equipment Setup
  • 3. Synthetic Protocols
  • 3.1. 8-Amino-2-quinolinecarboxylic Acid-Based Oligoamides
  • 3.1.1. Protocol 1: Bromination of Low-Loading Wang Resin
  • 3.1.2. Protocol 2: Loading of Wang-Bromide Resin
  • 3.1.3. Protocol 3: Fmoc Deprotection
  • 3.1.4. Protocol 4: Conversion of N-Fmoc Quinoline Carboxylic Acid Monomer 4 to the Corresponding Acid Chloride, 6
  • 3.1.5. Protocol 5: Coupling of N-Fmoc Quinoline Carboxylic Acid Chloride (6) to Resin Bound Amine (7)
  • 3.1.6. Protocol 6: Assessment of Coupling Completion Using a Modified DESC Test
  • 3.1.7. Protocol 7: Acetylation of the N-Terminal Aromatic Amine
  • 3.1.8. Protocol 8: Cleavage of Foldamer from the Resin
  • 3.2. 7-Amino-8-fluoro-2-quinolinecarboxylic Acid-Based Oligoamides
  • 3.3. Quinoline/a-Amino Acid Hybrid Oligoamides
  • 3.3.1. Protocol 9: Coupling of a-Amino Acid to the Quinoline Amine via In Situ Acid Chloride Formation
  • 3.3.2. Protocol 10: Acetylation of Aliphatic N-Terminal Amine
  • 4. Characterization of Foldamers
  • 4.1. 8-Amino-2-quinolinecarboxylic Acid-Based Oligoamides
  • 4.2. 7-Amino-8-fluoro-2-quinolinecarboxylic Acid-Based Oligoamides
  • 4.3. Quinoline/a-Amino Acid Hybrid Oligoamides
  • References
  • Chapter Fourteen: Conformational Restriction of Peptides Using Dithiol Bis-Alkylation
  • 1. Introduction
  • 1.1. Stabilizing Alpha-Helical Structures
  • 1.2. Stabilizing Nonhelical Structures
  • 2. Using Thiol Alkylation to Constrain Peptides
  • 2.1. Libraries of Peptides That Are Constrained Through Alkylated Cysteines
  • 2.2. Rational Design Using Cysteine Alkylation
  • 2.3. Thiol Bis-Alkylation Is Ideal for Constraining Epitopes into Diverse Conformations
  • 3. Protocols for Peptide Synthesis and Cross-Linking
  • 4. Applications, Tips, and Troubleshooting
  • 4.1. Applying Dithiol Bis-Alkylation for Constraining Loop Epitopes
  • 4.2. Monitoring Progression of Thiol Bis-Alkylation
  • 4.3. Reaction Scope and Versatility
  • 4.4. Investigating the Bis-Alkylation Mechanism
  • 4.5. Optimizing and Troubleshooting Bis-Alkylation Reactions
  • 4.5.1. Concentration
  • 4.5.2. Relative Amount of Linker
  • 4.5.3. Solvent Selection
  • 4.5.4. Presence of Reducing Agents
  • 4.5.5. pH Dependence
  • 5. Future Directions
  • References
  • Chapter Fifteen: Engineering Short Preorganized Peptide Sequences for Metal Ion Coordination: Copper(II) a Case Study
  • 1. Introduction
  • 1.1. Designing Short Preorganized Peptide Sequences for Cu(II) Coordination
  • 2. Design of Preorganized Peptidic Scaffolds for Metal Ion Coordination
  • 3. Synthesis and Characterization of the Peptidic Scaffolds
  • 3.1. Synthesis of Linear Scaffolds
  • 3.2. Synthesis of Cyclic Scaffolds
  • 4. Preparation of Analytical Stock Solutions
  • 4.1. Peptidic Scaffolds
  • 4.1.1. Trp/Tyr/Disulfide-Containing Scaffolds (Edelhoch, 1967
  • Gill Pace, Vajdos, Fee, Grimsley, & Gray, 1995)
  • 4.1.2. Cys-Containing Scaffolds (Ellman, 1959)
  • 4.1.3. Scaffolds with No Trp, Tyr, and Cys (Scopes, 1974)
  • 4.2. Metal Cation Stock Solutions
  • 4.3. Electrolyte Stock Solution
  • 4.4. Base Titrant Solution
  • 4.5. Acid Titrant Solution
  • 5. Study of the Metal Ion Coordination Properties
  • 5.1. Potentiometric Titrations: Determining Stability Constants and Speciation Diagrams
  • 5.1.1. Calibration of the pH Electrode
  • 5.1.2. Potentiometric Titrations
  • 5.1.3. Potentiometric Data Treatment
  • 5.2. Characterization of Major Species by Spectroscopy
  • 6. Concluding Remarks
  • Acknowledgments
  • References
  • Chapter Sixteen: De Novo Construction of Redox Active Proteins
  • 1. Introduction
  • 2. Sequence Selection: Binary Patterning
  • 3. Sequence Selection: Cofactor Self-Assembly
  • 4. Binding Heme Redox Cofactors
  • 5. Electron-Transfer Reactions of Heme Maquettes
  • 6. Binding Light-Activated Zn Tetrapyrrole Cofactors
  • 7. Iron-Sulfur Cluster Cofactors
  • 8. Redox Metal Binding Sites
  • 9. Flavin Cofactor Binding and Electron Transfer
  • 10. Translating Aqueous Redox Maquette Designs into Membranes
  • References
  • Chapter Seventeen: Design Strategies for Redox Active Metalloenzymes: Applications in Hydrogen Production
  • 1. Introduction
  • 2. Design of FeS Clusters
  • 2.1. Use of Natural Sequences
  • 2.2. Computational Design of Model Proteins
  • 2.3. Incorporation into Prestructured Proteins
  • 3. Design of [FeFe]-Hydrogenase Mimics
  • 3.1. Use of Peptide Scaffolds
  • 3.2. Use of Natural Sequences
  • 4. Design of Porphyrin Redox Sites
  • 4.1. Redesign of Natural Scaffolds
  • 4.2. Use of Peptide Scaffolds
  • 5. Conclusion and Future Outlook
  • References
  • Chapter Eighteen: Equilibrium Studies of Designed Metalloproteins
  • 1. Introduction
  • 2. Metalloprotein Thermodynamics
  • 2.1. The Influence of Protons on Metal-Ion Affinity
  • 2.2. The Thermodynamic Relationship Between Heme Electrochemistry and Affinity
  • 2.3. The Influence of Protons on Heme Electrochemistry
  • 2.4. The Thermodynamic Relationship Between Heme Electrochemistry and Protein Folding
  • 3. Essential Equilibrium Measurements
  • 3.1. Cofactor Binding Equilibria
  • 3.2. Protonation Equilibrium
  • 3.3. Electrochemical Equilibrium
  • 4. Thermodynamic Analysis of a Heme Binding Four Helix Bundle, [?7-His]2
  • 4.1. Heme Affinity
  • 4.2. Electrochemistry
  • 4.3. Proton Competition
  • 4.4. The PCET Event
  • 5. Experimental Procedures
  • 5.1. Protein Synthesis and Purification
  • 5.2. Equilibrium Measurements
  • 5.3. Kinetic Evaluation
  • 6. Summary
  • Acknowledgment
  • References
  • Chapter Nineteen: Reconstitution of Heme Enzymes with Artificial Metalloporphyrinoids
  • 1. Introduction
  • 2. Design and Synthesis of Artificial Metalloporphyrinoids
  • 2.1. Dianionic Porphyrinoid Ligand
  • 2.2. Trianionic Porphyrinoid Ligand
  • 2.3. Monoanionic Porphyrinoid Ligand
  • 3. Reconstitution of Hemoproteins
  • 3.1. Preparation of Apoproteins
  • 3.2. Common Protocols for Heme Protein Reconstitution with Artificial Metalloporphyrinoids
  • 3.3. Characterization of Reconstituted Proteins
  • 4. Representative Characteristics of Reconstituted Hemoproteins
  • 4.1. Peroxidase Activity of Reconstituted Myoglobin and HRP
  • 4.2. Hydroxylase Activity of Reconstituted Myoglobin
  • 4.3. Methionine Synthase Model
  • Acknowledgments
  • References
  • Chapter Twenty: Creation of a Thermally Tolerant Peroxidase
  • 1. Introduction
  • 2. Molecular Design for Cyt c552 to Acquire Peroxidase Activity
  • 3. Gene Constructs of Recombinant Wild Type and Mutant Cyt c552
  • 3.1. Materials
  • 3.2. Procedures
  • 4. Expression and Purification of the Recombinant Proteins
  • 4.1. Materials
  • 4.2. Procedures
  • 5. Thermal Stability of the Cyt c552 Mutants
  • 5.1. Materials
  • 5.2. Procedures
  • 6. Analysis of a Key Intermediate in the Peroxidase Reaction of Cyt c552 V49D/M69A Mutant
  • 6.1. Materials
  • 6.2. Procedures
  • 7. Effect of Trp45 on the Active Intermediate of Mutants
  • 7.1. Procedures
  • 7.1.1. Kinetic Measurement
  • 7.1.2. Measurement of Catalytic Activity
  • References
  • Chapter Twenty-One: Designing Covalently Linked Heterodimeric Four-Helix Bundles
  • 1. Introduction
  • 1.1. The Four-Helix Bundle: A Widespread Structural Motif
  • 1.2. Designing Functional Four-Helix Bundle Proteins
  • 2. Selection of the Best Docking Hotspot Given a Predefined Anchor Bolt
  • 2.1. Structure Preparation of the Target Protein
  • 2.2. Structure Preparation of the Linker
  • 2.3. Performing the Geometrical Parameter Calculations
  • 2.4. Data Analysis
  • 2.5. Generation of the Best Candidates for Click Reaction
  • 2.6. Evaluation of the Best Candidates for Synthesis
  • 3. Broadening the Hotspot and Linker Selections
  • 3.1. Structure Preparation of Any Target Protein
  • 3.2. Structure Preparation of the Linker Library
  • 3.3. Performing the Geometrical Parameter Calculations for Each Residue Pair
  • 3.4. Data Analysis
  • 3.5. Generation of the Best Candidates for the Identified Residue Pairs
  • 3.6. Evaluation of the Best Models Amenable for the Selected Linkers
  • 4. Concluding Remarks
  • Acknowledgments
  • References
  • Chapter Twenty-Two: Design of Heteronuclear Metalloenzymes
  • 1. Step 1: Computational Design of Heteronuclear Metal-Binding Sites in Mb or CcP
  • 1.1. Structure-Guided Design of Primary Ligands
  • 1.2. Design of Secondary Coordination Sphere Interactions
  • 1.3. Computational Analysis of the Design
  • 2. Step 2: Purification and Structural Characterization of Rationally Designed Proteins
  • 2.1. UV-Vis Spectroscopy
  • 2.1.1. UV-Vis Spectroscopic Titration of Nonheme Fe(II) in E-FeBMb
  • 2.2. Mass Spectrometry (MS)
  • 2.3. EPR
  • 2.4. X-Ray Crystallography
  • 3. Step 3: Functional Characterization of Designed Heterobinuclear Metalloenzymes
  • 3.1. Activity Assays
  • 3.1.1. Catalytic Efficiency of the Reaction
  • 3.1.2. Product Selectivity
  • 3.1.3. Turnover Numbers
  • 3.2. Redox Potential Measurement
  • 3.3. Kinetic and Mechanistic Studies
  • 3.4. Capture and Characterization of Reaction Intermediates
  • 4. Step 4: Further Improvement of Designed Heteronuclear Metalloenzymes: Case Studies
  • 4.1. Improving ET to the Heme Center
  • 4.2. Tuning the Heme Reduction Potential
  • 4.3. Increasing the Binding Affinity of Mn2+ in MnCcP
  • 5. Conclusions
  • Acknowledgments
  • References
  • Chapter Twenty-Three: Periplasmic Screening for Artificial Metalloenzymes
  • 1. Introduction
  • 2. Anchoring Strategies
  • 3. Increasing the Throughput
  • 4. Advantages of Periplasmic Screening
  • 5. Periplasmic Screening for ArMs
  • 5.1. Choice of Expression Strain and Translocation Strategy
  • 5.2. Establishment of a Screening Workflow
  • 5.2.1. Library Generation
  • 5.2.2. Cultivation and Expression
  • 5.2.3. Cofactor Uptake and ArM Assembly
  • 5.2.4. Activity Assay and Mutant Retrieval
  • 6. Summary
  • References
  • Chapter Twenty-Four: De Novo Designed Imaging Agents Based on Lanthanide Peptides Complexes
  • 1. Introduction
  • 2. Lanthanide Coordination Chemistry
  • 3. Lanthanide Imaging Agents
  • 4. Lanthanide Peptides and Proteins
  • 4.1. Inspired by Native Ca(II)-Binding Loops
  • 4.2. Other Lanthanide-Binding Sites
  • 5. Strategies for Designing CCs for Lanthanide Coordination
  • 5.1. CC Design
  • 5.2. Lanthanide-Binding Site Design
  • 5.2.1. At the Helical Interface
  • 5.2.2. Within the Hydrophobic Core
  • 5.3. Controlling Water Coordination and Access
  • 5.3.1. Cavity
  • 5.3.2. Enhanced Steric Bulk
  • 5.3.3. Location Within the CC
  • 5.4. Lanthanide Selectivity
  • 5.5. Controlling Stability
  • 5.5.1. Peptide Folding
  • 5.5.2. Proteolytic Stability
  • 5.5.3. Gadolinium Dissociation Constant
  • 5.6. MRI Efficiency of Gadolinium CCs
  • 6. Conclusions and Perspectives
  • Acknowledgments
  • References
  • Chapter Twenty-Five: Peptide Binding for Bio-Based Nanomaterials
  • 1. Introduction
  • 1.1. General Opening Remarks
  • 1.2. Biocombinatorial Approaches to Isolate Peptides with Material Affinity
  • 1.3. Peptide-Based Approaches for NP Production
  • 1.4. Advantages and Limitations of Peptides for NP Production
  • 2. Materials
  • 2.1. Materials for Peptide Synthesis
  • 2.2. Materials for Peptide Purification
  • 2.3. Materials for Peptide-Capped NP Synthesis
  • 2.4. Materials for PDF Analysis
  • 3. Methods
  • 3.1. Peptide Synthesis
  • 3.1.1. Solutions Required for Peptide Synthesis
  • 3.1.2. Protocols for Automated Synthesis
  • 3.1.3. Cleavage from Resin
  • 3.1.4. Purification via Reverse-Phase HPLC
  • 3.2. Peptide-Capped NP Synthesis
  • 3.2.1. Solutions Required for Peptide-Capped NP Synthesis
  • 3.2.2. Protocols for NP Synthesis
  • 3.2.3. Characterization of Peptide-Capped NPs
  • 3.3. Methods for PDF Analysis
  • 3.3.1. Sample Preparation
  • 3.3.2. HE-XRD Data Acquisition
  • 3.3.2.1. Sample Preparation
  • 3.3.2.2. Calibrating the Sample to Detector Distance
  • 3.3.2.3. Setting Acquisition Times
  • 3.3.2.4. Data Acquisition
  • 3.3.3. HE-XRD Pattern Conversion to PDFs
  • 3.3.3.1. HE-XRD Integration
  • 3.3.3.2. Calculating PDFs
  • 3.3.4. Structural Modeling
  • 4. Conclusions
  • Acknowledgments
  • References
  • Author Index
  • Subject Index
  • Color Plate
  • Back Cover

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