Protein Design and the Development of New Therapeutics and Vaccines

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I. Advances in the Structural Analysis of Proteins.- 1 Dynamic Processes in Proteins by X-Ray Diffraction.- 1. Dynamic Processes in Proteins.- 2. Enzyme Substrate Complexes by X-Ray Diffraction.- 3. Laue Diffraction.- 4. Reaction Synchronization.- References.- 2 Mass Spectrometric Methods for Determination of the Structure of Peptides and Proteins.- 1. Introduction.- 2. High-Mass Mass Spectrometry.- 3. Tandem Mass Spectrometry.- 3.1. Sequencing of Proteins.- 3.2. Increasing Sensitivity by Using an Array Detector.- 3.3. Computer-Aided Interpretation of CID Spectra of Peptides.- 4. Conclusion.- References.- II. Protein Folding and Stability.- 3 Protein Folding and Stability.- 1. Goals.- 2. Folding of Fragments.- 3. Trapping Folding Intermediates.- References.- 4 Genetic Analysis of Polypeptide Chain Folding and Misfolding in Vivo.- 1. Introduction.- 2. The Protein Folding Grammar.- 3. Intracellular Intermediates in Chain Folding and Assembly.- 4. Temperature-Sensitive Folding Mutations.- 5. Nonsense Mutations.- 6. Intracellular Aggregation and Inclusion Body Formation.- 7. Kinetics of Aggregate Formation.- 8. Isolation of Second-Site Suppressors of Folding Mutants.- 9. The Suppressing Amino Acid Substitutions Act at the Level of Polypeptide Chain Maturation.- 10. The Suppressors Act in the Folding of the Single Chain, Not during Chain—Chain Association.- 11. The Suppressors Increase the Efficiency of Folding of the Wild-Type Chain.- References.- 5 Unfolding and Inactivation: Genetic and Chemical Approaches to the Stabilization of T4 Lysozyme and Human Interferon Gamma against Irreversible Thermal Denaturation.- 1. Introduction.- 2. T4 Lysozyme.- 2.1. Sulfhydryl Oxidation.- 2.2. Thiol/Disulfide Interchange.- 2.3. Adsorption.- 2.4. Aggregation.- 2.5. Chemical Inactivation.- 2.6. Conclusions from T4 Lysozyme Experiments.- 3. Interferon-?.- 3.1. In Vitro Experiments.- 3.2. Genetic Experiments.- 4. General Conclusions.- 4.1. Modes of Thermal-Irreversible Denaturation.- 4.2. Strategies for Stabilizing Proteins.- References.- III. Principles of Receptor Design and Regulation.- 6 Protein—Tyrosine Kinases and Their Substrates: Old Friends and New Faces.- 1. Introduction.- 2. Isolation of Novel Protein—Tyrosine Kinases.- 3. Regulation of Protein—Tyrosine Kinase Activity: Phosphorylation of pp60c-src.- 4. Protein—Tyrosine Kinase Substrates.- References.- IV. The Guanine Nucleotide Binding Protein Family.- 7 G Proteins: A Family of Signal-Transducing Molecules.- 1. The Family of G Proteins.- 2. Distribution of G Protein ? Subunits.- 3. Regulation of ?o Levels.- 4. G Proteins as Bifurcating Signals.- 5. Organizing Principles.- References.- 8 Model of Signal Transduction by G Proteins: Roles of a Subunits and ?? Dimers in Regulation of Ionic Channels and Adenylyl Cyclase.- 1. Introduction.- 2. Mechanisms of Receptor-Mediated Activation of G Proteins.- 3. Which G-Protein Subunit Mediates the Effect of Receptors?.- 3.1. Regulation of Ionic Channels: ? or ??, and What Does Each Do to Regulation of Channel Activity?.- 3.2. Inhibitory Regulation of Adenylyl Cyclase: ?i or ???.- 3.3. What Is the Role of ?? Dimers?.- References.- 9 Structural Homologies in G-Binding Proteins.- 1. Introduction.- 2. Structural Homology in Three Dimensions.- 2.1. Elongation Factor Tu (EF-Tu).- 2.2. ras Protein p21.- 3. Sequence Homology.- 3.1. Overall Homology.- 3.2. Consensus Sequences.- 4. Homology and Functional Similarity.- References.- V. Modeling and Structure Prediction in Macromolecules.- 10 Knowledge-Based Protein Modeling and the Design of Novel Molecules.- 1. Introduction.- 2. Learning by Comparison of Structures.- 2.1. Superposition of Structures.- 2.2. Comparison of Properties and Relations.- 2.3. Clustering and Tree Construction.- 3. Rules for Tertiary Templates and Key Residues, and Their Comparison with a Sequence of an Unknown.- 3.1 Consensus Sequences for Framework Regions.- 3.2. Key Residues from Comparisons of Properties and Relations.- 3.3. Characteristic Sequences from Features of Tertiary Structure.- 4. Generation of a Model from a Sequence of an Unknown Using Rules from Comparison of Structures.- 4.1. Construction of the Model by Assembly of Rigid Groups in Three Dimensions.- 4.2. Construction of a Model Using Optimization Techniques.- 4.3. Refinement of the Model.- 5. Applications to Design of Novel Molecules.- 5.1. Receptor-Based Drug Design.- 5.2. Site-Directed Mutagenesis.- 5.3. Chimeric Molecules.- 5.4. Ab Initio Protein Design.- References.- 11 Computer Simulation Methods as Applied to Site-Specific Mutations.- 1. Introduction.- 2. Methodology.- 3. Applications of Molecular Dynamics/Free-Energy Calculations to Site-Specific Mutagenesis.- 3.1. Structure Correlation with X-Ray Observations.- 3.2. Free Energies of Solvation and Ligand Binding.- 3.3. Free Energies of Binding and Catalysis of Site-Specific Mutants.- 3.4. Free Energies of Protein Stability.- 4. Summary and Critique.- References.- 12 Dihydrofolate Reductase: A Paradigm for Drug Design.- 1. Introduction.- 2. Structure and Kinetics of Dihydrofolate Reductase from E. coli.- 2.1. Structure.- 2.2. Kinetics.- 3. Mutagenesis Studies.- 3.1. Ligand Binding and Catalytic Turnover.- 3.2. Long-Range Effects.- 3.3. Nonconserved Residues.- 4. Conclusions.- References.- VI. Protein Engineering and Enzyme Design.- 13 Chemical Approaches to Protein Engineering.- 1. Introduction.- 2. Types of Chemical Approach.- 2.1. Total Synthesis.- 2.2. Side-Chain Modification.- 3. Semisynthesis.- 3.1. Noncovalent Semisynthesis.- 3.2. Covalent Semisynthesis: Disulfide Bridges.- 3.3. Covalent Semisynthesis: The Peptide Bond.- 4. Recent Advances in Knowledge Achieved with Semisynthetic Proteins.- 4.1. Insulin.- 4.2. Other Hormones.- 4.3. Cytochrome c.- 4.4. Hemoglobins.- 4.5. Phospholipase A2.- 5. Linkages Other than by Peptide Bonds.- 5.1. Linkages at the Amino Terminus.- 5.2. Linkages at the Carboxyl Terminus.- 6. Conclusions.- References.- 14 New Engineered Proteins for Use in Therapy and Vaccine Design.- 1. Introduction.- 2. New Generation of Factor VIII Molecules for the Treatment of Hemophilia A.- 3. Hirudin: Variants with Improved Antithrombic Activity.- 4. ?1-Antitrypsin.- 5. HIV Envelope Protein: Improved Antigenicity by Cleavage Site Removal.- References.- 15 Receptor-Based Design of Dihydrofolate Reductase Inhibitors.- 1. Introduction.- 2. The Enzyme.- 2.1. Role in Cellular Metabolism.- 2.2. Primary Structure.- 2.3. Three-Dimensional Structure.- 3. Enzyme—Inhibitor Interactions.- 3.1. Overview.- 3.2. DHFR—Methotrexate Complexes.- 3.3. DHFR—Trimethoprim Complexes.- 4. Examples of Inhibitor Design.- 4.1. Trimethoprim Analogs with Acidic Substituents.- 4.2. Conformationally Restricted Analogs of Trimethoprim.- 5. Conclusion.- References.- VII. Macromolecules and Targeted Drug Delivery.- 16 Control of the Biological Dispersion of Therapeutic Proteins.- 1. Introduction.- 2. Therapeutic Proteins.- 2.1. Type.- 2.2. Physical and Chemical Properties.- 3. Clinical Use.- 3.1. Site and Mode of Action.- 3.2. Endocrine-Like and Para-/Autocrine-Like Mediators.- 4. Protectants.- 5. Protein (Re)glycosylation.- 5.1. Oligosaccharide Variability and Recognition.- 5.2. Biotechnological Processes.- 5.3. Protein Remodeling.- 5.4. Enzyme-Storage Diseases.- 5.5. Deglycosylation.- 6. Protein Hybrids.- 6.1. Cell Processing.- 6.2. Gene-Fusion Hybrids.- 6.3. Synthetically Linked Hybrid Conjugates.- 6.4. Antibodies.- 7. Administration.- 7.1. Route, Rate, and Frequency of Input.- 7.2. Parenteral/Interstitial Administration.- 7.3. Gastrointestinal Tract.- 7.4. Nasal and Buccal Mucosae.- 8. Concluding Remarks.- References.- 17 Antibody-Mediated Drug Delivery.- 1. Introduction.- 2. Global and Regional Pharmacokinetics.- 3. Microvascular Transport.- 4. Tissue Level: The Percolation Problem.- 5. Percolation Calculations and the Binding Site Barrier.- 6. Concluding Remarks.- References.- 18 Semisynthetic Catalytic Antibodies.- 1. Design Considerations.- 2. Synthesis of Affinity Labels.- 3. Antibody Modification.- 3.1. Affinity Labeling.- 3.2. Cleavage of Labels and Isolation of Stable S-Thiopyridyl Adducts.- 4. Determination of Modified Site.- 5. Ester Cleavage Using Thiol-Derivatized Antibody.- 5.1. Synthesis of Substrates.- 5.2. Ester Cleavage Assays.- 5.3. Substrate Specificity and Enantioselectivity.- 6. Derivatization with Imidazole.- 7. Derivatization with a Spectroscopic Probe.- 8. Prospects.- References.- VIII. Strategies for Directed Mutagenesis of Proteins.- 19 Engineering Novel, Prolonged-Acting Insulins.- 1. Introduction.- 1.1. Background and Aims.- 1.2. Rationale of Design and Mode of Action of Prolonged-Acting, Soluble Insulins.- 2. Synthesis of Insulin Analogs.- 2.1. The Tryptic Transpeptidation Reaction.- 2.2. Single-Chain Des-(B30) Insulins.- 2.3. Biosynthesis in Yeast.- 2.4. Substitutions that Increase pI.- 2.5. Substitutions that Stabilize Insulin.- 3. Biological Evaluation.- 3.1. Free Fat Cell Bioassay.- 3.2. Mouse Blood Glucose Assay.- 3.3. Index of Prolongation.- 3.4. Absorption Kinetics in Pigs Using External ?-Counting.- 4. Crystallization Techniques.- 4.1. Micro Reaction Chamber.- 4.2. Hanging-Drop Crystallization.- 4.3. Crystallization In Vivo.- 5. Results and Discussion.- 5.1. Substitutions that Enhance the Prolonged Action.- 5.2. Substitutions without Significant Effect on Absorption.- 5.3. Substitutions that Enhance Stability.- 5.4. Failure of Expression of ProA21.- 5.5. Galenic Factors Influencing Absorption Kinetics.- 6. Preclinical Studies and Considerations.- 6.1. Reproducibility of Absorption.- 6.2. Calculation of Variations in Depots and Blood Levels of Basal Insulin in the Steady State.- 6.3. Potency and Units.- 6.4. Immunogenicity.- 7. Summary.- References.- IX. Design of Novel Antiviral Agents and Vaccines.- 20 Studies on the Structure and Function of Ubiquitin.- 1. Introduction.- 2. Synthesis and Expression of Ubiquitin and Mutant Genes in Yeast and Escherichia coli.- 3. Disulfide Mutants.- 4. Carboxy Terminal Peptides.- 5. Conclusions.- References.- 21 Recognition at Membrane Surfaces: Influenza HA and Human HLA.- 1. Introduction.- 2. HA—Cell Receptor Interaction.- 2.1. Nuclear Magnetic Resonance and Solution Studies of Binding.- 2.2. Antigenic Variation and Inhibitor Design.- 3. Virus Entry and Membrane Fusion.- 4. Human Histocompatibility Antigens.- 4.1. The Structure of HLA-A2.- 4.2. Peptide Binding.- 4.3. The Structure of HLA-A28.- 4.4. Modeling Class II Histocompatibility Antigens.- References.