Phosphatases / edited by Karen N. Allen.

Format:

Book

Contributor:

Allen, Karen N., editor.

Series:

Methods in enzymology ; 0076-6879 Volume 607.

Methods in enzymology ; Volume 607

Language:

English

Subjects (All):

Phosphatases.

Physical Description:

1 online resource (442 pages).

Place of Publication:

Cambridge, MA : Academic Press is an imprint of Elsevier, [2018]

Summary:

Methods in Enzymology, Volume 607: Phosphatases, the latest release in this ongoing series, highlights new advances in the field as detailed by an international board of authors. This latest release includes chapters on Empirical Valence Bond Simulations of the Evolution of Enzyme Function, QM/MM Free Energy and Kinetic Isotope Analysis of Phosphoryl Transfer in Enzymes, the Structural, Mechanism and Evolution of Phosphatases, How to Define Rapid Motions in Pumping Pyrophosphatases, The Evolution of K+-Independence in Pyrophosphatases, the Crystallization of Michaelis, Intermediate and Inhibited Complexes in Phosphatases, and an Investigation of Nucleotide Loading and Effector Binding of K-Ras.- Provides the authority and expertise of leading contributors from an international board of authors- Presents the latest release in the Methods in Enzymology series- Updated release includes the latest information on phosphatases

Contents:

Front Cover

Phosphatases

Copyright

Contents

Contributors

Preface

Section I: Computational Analysis of Enzymatic Phosphoryl Transfer

Chapter One: Empirical Valence Bond Simulations of Organophosphate Hydrolysis: Theory and Practice

1. Introduction

2. The Empirical Valence Bond Approach

3. Empirical Valence Bond Simulations in Practice

3.1. Obtaining the Relevant Software

3.2. System Preparation

3.2.1. Structure Preparation

3.2.2. Assigning Correct Protonation States

3.2.3. Force-Field Parameterization

3.2.4. Substrate Placement in the Active Site

3.2.5. System Solvation and Topology Generation

3.2.6. Creating an EVB Parameter File

3.3. System Equilibration

3.3.1. Removing Steric Clashes

3.3.2. Thermal Equilibration

3.4. Simulating Chemical Reactions

3.4.1. Sampling on the Mapping Potential

3.4.2. Obtaining the EVB Free-Energy Profiles

3.4.3. Calibrating the EVB Potential

4. Empirical Valence Bond Simulations: Sample Results

5. Technical Considerations

5.1. Convergence and Reproducibility

5.2. Chemical and Conformational Coordinates

5.3. Over the Mountain Pass or Rappelling Into Valleys?

5.4. Troubleshooting

5.4.1. System ``Explosions"

5.4.2. EVB Mapping Failures and Physically Unreasonable Results

5.4.3. ``Rough" Free-Energy Profiles

5.4.4. Large Variations in the Free-Energy Results

6. Overview and Conclusions

References

Chapter Two: Analysis of Phosphoryl-Transfer Enzymes with QM/MM Free Energy Simulations

2. Background on Computational Methods

2.1. QM/MM Potential Function

2.1.1. Semiempirical QM/MM Methods

2.1.2. Ab Initio and DFT QM/MM Methods

2.2. Free Energy Computations With QM/MM Potentials

2.2.1. Direct Sampling: Metadynamics and String Methods.

2.2.2. Minimum Free Energy Path Approach for Ab Initio QM/MM

2.2.3. Multilevel QM/MM Free Energy Calculations

2.3. Other Experimental Observables

3. Case Studies

3.1. Alkaline Phosphatase: Phosphoryl-Transfer Transition State

3.1.1. Nature of Transition State and Free Energy Relationships

3.1.2. Nature of Transition State and Kinetic Isotope Effects

3.1.3. Remaining Questions and Methodological Challenges

3.2. Myosin: Regulation of the Timing of ATP Hydrolysis

3.2.1. Multiple Reaction Pathways

3.2.2. Impact of Global Structural Transitions

3.2.3. Remaining Questions and Methodological Challenges

4. Concluding Remarks

Acknowledgments

Section II: Structure, Mechanism and Evolution of Phosphatases

Chapter Three: Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET

1.1. mPPase Structure and Mechanism

1.2. Defining Dynamic Motions in mPPases by FRET

2. Combining Experimental and Computational Methods for Optimizing the Attachment of Fluorescent Dyes for Single-Molecule Fret

2.1. Choosing Positions for Mutagenesis From Sequence and Structure

2.1.1. Equipment and Software

2.1.2. Procedure

2.1.3. Notes

2.2. Expression, Harvest, and S. cerevisiae Membrane Preparation

2.2.1. Equipment and Consumables

2.2.2. Buffers and Reagents

2.2.3. Procedure

2.2.4. Notes

2.3. Solubilization and Purification of TmPPase Cysteine Variants

2.3.1. Equipment and Consumables

2.3.2. Buffers and Reagents

2.3.3. Procedure

2.3.4. Notes

2.4. Activity of Cysteine Variants

2.4.1. Equipment

2.4.2. Buffers and Reagents

2.4.3. Procedure

2.4.4. Notes

2.5. Labeling of TmPPase Cysteine Variants

2.5.1. Equipment

2.5.2. Buffers and Reagents

2.5.3. Procedure.

2.6. Reconstitution of Labeled TmPPase Into Liposomes

2.6.1. Equipment and Consumables

2.6.2. Buffers and Reagents

2.6.3. Procedure

2.7. All-Atom Molecular Dynamics Simulations

2.7.1. Equipment and Software

2.7.2. Procedure

2.7.3. Notes

2.8. Computational Methods to Account for Dye Flexibility

2.8.1. Equipment

2.8.2. Procedure

2.8.3. Notes

2.9. Single-Molecule FRET Using Alternating Laser Excitation

2.9.1. Equipment

2.9.2. Procedure

2.9.3. Notes

3. Summary and Conclusions

3.1. Nonfunctional Cysteine Variants of Loop 5-6

3.2. Improved Strategy for Selecting Sites for Cysteine Mutagenesis

3.3. Using MD Simulations and Computational Modeling to Assist Selection

3.4. Labeling TmPPase Double Variant and Observing smFRET in the Presence of Substrate or Inhibitors

Acknowledgment

Chapter Four: A Simple Strategy to Determine the Dependence of Membrane-Bound Pyrophosphatases on K+ as a Cofactor

1.1. Distribution, Biological Role, and Significance of mPPases

1.2. The Evolution and Structure of mPPases

1.3. Determination of K+ Dependence in mPPases

2. Preliminary Sequence-Based Determination of K+ Dependency

2.2. PaPPase Expression in Saccharomyces cerevisiae and Membrane Extraction

2.3. Solubilization and Purification of PaPPase Using the ``Hot-Solve" Method

2.4. Quantitative Phosphate Assay

2.5. Data Analysis

2.5.1. Procedure

2.5.2. Notes

3. Summary and Conclusion.

Acknowledgments

Chapter Five: Crystallization of Liganded Phosphatases in the HAD Superfamily

2. Substrate and Product Complexes

2.1. Substrate Soaking

2.2. Ligand-Soaking Experiments

2.2.1. Equipment

2.2.4. Notes and Examples

2.3. Mutation of the Catalytic Residue(s)

2.4. Exchange Metals

2.5. Phosphate Binding

3. Ground-State, Intermediate, and Transition-State Mimic Complexes

3.1. Intermediate-Bound Complexes

3.2. Ground-State Mimic Complexes

3.3. Transition-State Mimic Complexes

3.4. Activation of Sodium Orthovanadate

3.4.1. Equipment

3.4.2. Buffers and Reagents

3.4.3. Procedure

3.4.4. Notes

4. Inhibitors

4.1. Substrate Mimics as Inhibitors

4.2. Protein and Crystal Stability in the Presence of Inhibitors/Solvents

4.2.1. Determining Protein Stability

4.2.1.1. Equipment

4.2.1.2. Buffers and Reagents

4.2.1.3. Procedure

4.2.1.4. Notes

4.2.2. Optimizing Crystal Stability

4.2.2.1. Equipment

4.2.2.2. Buffers and Reagents

4.2.2.3. Procedure

4.2.2.4. Notes

5. Summary and Conclusion

Section III: Substrate Specificity and Enzyme Conformational States

Chapter Six: Structure, Mechanism, and Substrate Profiles of the Trinuclear Metallophosphatases from the Amidohydrolase S ...

2. L-Histidinol Phosphate Phosphatase (Ghodge, Fedorov, et al., 2013)

3. Cyclic Phosphate Dihydrolase (Ghodge, Cummings, Williams, & Raushel, 2013)

4. 3′,5′-Adenosine Bisphosphate-3′-Phosphatase (Cummings et al., 2014)

5. RNase AM (Ghodge & Raushel, 2015)

Further Reading

Chapter Seven: Affinities and Comparisons of Enzyme States by Principal Component Analysis of NMR Spectra, Automated usin ...

1. Introduction.

2. Insights Available Semiautomatically using TREND NMR Software

2.1. PCA Comparison of Multiple Enzyme Samples

2.2. PCA Tracking of Changes to a Single Enzyme Sample

3. Capabilities of TREND NMR Suite with Examples

3.1. Binding Isotherms and Affinities Directly From Unassigned NMR Spectra

3.1.1. Titration With NMR Peaks That Disappear and Reappear (Slow Exchange)

3.1.2. Titration With Nonlinear Shifts and Broadening of Peaks (Intermediate Exchange)

3.2. Quick Comparison of Enzyme NMR Spectra and Conformational States

3.3. 1D Conformational Ordering of Enzyme Conformational Equilibria

4. NMR Data

4.1. Considerations and Forms of NMR Data

4.2. Choices of Format

5. Layout and Usage of TREND NMR Software

5.1. Platforms and Installation

5.2. TREND NMR and TRENDanalysis Functions

5.3. Procedure for Measuring Affinity From a Titration

5.4. Procedures for Statistical NMR Comparisons of Enzyme States

5.4.1. Steps Specific to CONCISE Ordering of States

5.4.2. Alternate Steps for Further Resolution of Enzyme States

6. Conclusions

Chapter Eight: Assessment and Impacts of Phosphorylation on Protein Flexibility of the α-d-Phosphohexomutases

2. Assessment and Modulation of the Phosphorylation State of the Catalytic P-Ser

2.1. Electrospray Ionization Mass Spectrometry

2.1.1. Equipment

2.1.2. Buffers and Reagents

2.1.3. Procedure

2.1.4. Notes

2.2. Reagents and Variables to Promote/Remove Modification of the P-Ser

3. Straightforward Assays to Characterize Differences between the Phosphorylated and Unphosphorylated Enzyme States

3.1. Limited Proteolysis

3.1.1. Equipment

3.1.2. Buffers and Reagents

3.1.3. Procedure

3.1.4. Notes.

3.2. Binding of the Fluorescent Dye ANS.

Notes:

Description based on print version record.

ISBN:

0-12-813882-3

0-12-813881-5