Protein Engineering

Protein Engineering

Approaches to the Manipulation of Protein Folding

1st Edition - March 26, 1990

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  • Editor: Saran A. Narang
  • eBook ISBN: 9781483161280

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Protein Engineering: Approaches to the Manipulation of Protein Folding outlines the complexity of the protein-folding problem and the potential of using genetic tools which, in combination with physical techniques, are expected to shed new light. The book begins with an overview of the basic concepts of protein folding, along with prediction methods and protein-folding models. Separate chapters cover experimental approaches to in vitro protein folding; general approaches used to characterize the folding reaction, equilibrium and kinetic experiments; and strategies employed to elucidate structure/function relationships in proteins of unknown tertiary structure. Subsequent chapters cover the structural and functional features of the HIV envelope protein; x-ray diffraction of proteins; application of Fourier transform infrared (FT-IR) spectroscopy to probe the secondary structure and orientation of membrane-associated proteins; and fluorescence measurements of proteins. The final chapters discuss nuclear magnetic resonance studies of proteins and the potential of the synthetic gene approach applied to the problem of protein folding.

Table of Contents

  • Preface


    1. Theories and Simulation of Protein Folding

    1.1 Basic Concepts

    1.2 Prediction Methods

    1.3 Protein-Folding Models

    1.4 Future Directions


    2. Experimental Approaches to Protein Folding

    3. Site-Directed Mutagenesis and Its Application to Protein Folding

    3.1 Principles and Questions

    3.2 Experimental Strategies

    3.3 Experimental Methods

    3.4 Analysis of Data from Mutant Proteins

    3.5 Experimental Results and Discussion

    3.6 Future Applications

    3.7 Conclusions


    4. The Dissection and Engineering of Sites That Affect the Activity of an Enzyme of Unknown Structure

    4.1 Strategies for Relating Protein Structure to Function

    4.2 Application to an Aminoacyl-Transfer RNA Synthetase

    4.3 Potential Engineering of Sites That Affect the Activity of Alanyl-Transfer RNA Synthetase

    4.4 Development and Testing of Structural Models


    5. Structural and Functional Features of the HIV Envelope Glycoprotein and Considerations for Vaccine Development

    5.1 The Envelope of HIV

    5.2 Mechanisms of Immune Attack on HIV

    5.3 Map of Immunologic and Functional Domains on the Envelope Glycoprotein

    5.4 Design of Vaccine Candidates


    6. Crystallographic Determination of Protein Structure

    6.1 Single-Crystal Diffraction

    6.2 Protein Crystallization

    6.3 Diffraction from Single Crystals

    6.4 Phase Determination

    6.5 Data Collection

    6.6 Fitting and Refinement

    6.7 Recent Advances in Protein X-Ray Crystallography

    6.8 Other Diffraction Techniques

    6.9 Conclusion


    7. The Conformation of Proteins and Peptides in a Membrane Environment: An Infrared Spectroscopic Approach

    7.1 Infrared Spectra and Protein Secondary Structure

    7.2 Polarized Infrared Spectra and the Orientation of Membrane-Protein Secondary Structures

    7.3 Studies with Native Membrane Proteins

    7.4 Studies with Membrane-Interacting Proteins and Peptides


    8. Application of Laser-Based Fluorescence to Study Protein Structure and Dynamics

    8.1 The Fluorescence Process

    8.2 Information from Fluorescence

    8.3 Time-Resolved Fluorescence

    8.4 Examples of Protein Fluorescence

    8.5 Fluorescence Anisotropy Decay

    8.6 Concluding Remarks


    9. Protein Structure Determination by Nuclear Magnetic Resonance Spectroscopy

    9.1 The Basic cH-NMR Experiment

    9.2 Two-Dimensional NMR Spectroscopy

    9.3 Sequential Resonance Assignments and Secondary Structure

    9.4 Calculation of Three-Dimensional Structures

    9.5 Quality of Structures and Comparison with X-Ray

    9.6 Structures of Ligands Bound to Proteins

    9.7 Future Trends in 1H-NMR

    9.8 Less-Sensitive Nuclei

    9.9 Theoretical Considerations

    9.10 13C-NMR Studies

    9.11 15N-NMR Studies

    9.12 31P-NMR Studies

    9.13 2H-NMR Studies

    9.14 Future Trends in Multinuclear NMR


    10. Synthetic Transposons

    10.1 Bacteriophage Mu Transposon

    10.2 Synthesis and Cloning of Mu-Ends DNA

    10.3 Transposition Assay

    10.4 Synthesis of Multiple Endonuclease Sites Between the Mu-Ends DNA

    10.5 In Vitro Transposition of Bacteriophage Mu

    10.6 Product of In Vitro Transposition Reaction

    10.7 Mechanism of Transposition of Bacteriophage Mu

    10.8 Perspective


    11. Redesigning Genes

    11.1 Problem of Protein Folding

    11.2 Synthetic Gene Approach to Protein Folding

    11.3 Gene Assembly

    11.4 Redesign and Synthesis of DNA Coding for T4-Lysozyme

    11.5 Hybrid-Gene Synthesis

    11.6 Construction of Libraries of Mutant Genes

    11.7 Site-Directed Mutagenesis

    11.8 Redesigning Antibodies

    11.9 Synthesis and Expression of Active Antibody Fragments in E. coli

    11.10 Humanization of Antibodies

    11.11 Chimeric Antibodies

    11.12 Single-Chain Antibodies

    11.13 De Novo Design of Protein and Its Synthesis by DNA Approach

    11.14 Future Direction




Product details

  • No. of pages: 284
  • Language: English
  • Copyright: © Butterworth-Heinemann 1990
  • Published: March 26, 1990
  • Imprint: Butterworth-Heinemann
  • eBook ISBN: 9781483161280

About the Editor

Saran A. Narang

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