Protein Engineering

Preface

Introduction


1. Theories and Simulation of Protein Folding


1.1 Basic Concepts


1.2 Prediction Methods


1.3 Protein-Folding Models


1.4 Future Directions

References


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

References


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

References


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

References


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

References


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

References


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

References


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

References


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

References


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

References

Epilogue

Index