Protein Engineering - 1st Edition - ISBN: 9780409901160, 9781483161280

Protein Engineering

1st Edition

Approaches to the Manipulation of Protein Folding

Editors: Saran A. Narang
eBook ISBN: 9781483161280
Imprint: Butterworth-Heinemann
Published Date: 26th March 1990
Page Count: 284
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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



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





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About the Editor

Saran A. Narang

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