Contents Abbreviations XV Prologue XVII fane S. Richardson 1 An Introduction to Protein Engineering 1 Thomas }. Graddis, Dale L. Oxender 1.1 Protein Engineering is a New Addition to the Biotechnology Revolution 1 1.2 Proteins Consist of Several Structural Elements 2 1.3 Technologies that Enable the Development of Protein Engineering . 3 Recombinant DNA and Genetic Engineering Technologies 4 — Protein Purifi- cation Plays a Role in the Cycle of Protein Engineering 8 — Functional Analy- sis is Used to Evaluate Structural Changes of Engineered Proteins 11 - Struc- tural Analysis of Proteins is Essential 12 - The Use of X-Ray Crystallography in the Structural Analysis of Proteins 12 — Nuclear Magnetic Resonance Spectroscopy is an Emerging Technology 13 — The Use of Design and Predic- tion in Protein Engineering 14 — De novo Design of Proteins is an Important Long-Term Goal 15 — Knowledge-Based Structural Prediction is Used to Model Proteins of Unknown Structure 16 1.4 There are Currently Several Bottlenecks to the Field of Protein Engi- neering 17 The Rules Governing Protein Folding are Complex 18 — A Few Recognized Steps in Protein Folding are Beginning to Emerge 19 — Chaperones are Pro- teins that Assist the Folding of Other Proteins 19 - Understanding the Struc- ture-Function Relationship is Central to Protein Engineering 20 - Computa- tional Chemistry is Finding Increasing Utility 20 — Data Base Management and Computer Graphics are Useful Tools 21 — Choosing a Prptein Expression System is Often an Empirical Science 22 — Advantages and Disadvantages of Protein Expression Systems 23 1.5 A Progress Report in Protein Engineering Includes Many Exciting Topics . 24 Attempts to Increase Protein Stability have Met with Outstanding Success 25 - Altering Enzyme Catalytic Activity has been Achieved in a Number of VIII Contents Systems 27 - Enzyme Specificity and Molecular Recognition are Fundamental to Enzyme Mechanism 28 — Peptide Hormones are Promising Targets for Pro- tein Engineering 30 — Significant Effort has been Applied to the Engineering of Antibodies 32 — Humanized Antibodies are Providing Important Thera- peutic Agents 32 - Catalytic Antibodies May Become Important Tools for Syn- thetic Chemists 33 — Random Libraries Represent a Semi-Rational Approach to Protein Engineering 33 — Synthetic Peptide Libraries Provide a Systematic Alteration of Peptide Sequence 34 — Combinatorial Libraries Generate Nu- merous Antibodies with Varied Binding Activity 35 — Phage Display Libraries Permit the Functional Screening of Vast Numbers of Protein Sequences 35 — The Future of Nanotechnology may Utilize DNA Structural Engineering 37 1.6 The Application of Protein Engineering to Agricultural, Industrial, and Medical Arenas has Produced Useful Products 38 Agricultural Applications 38 — Industrial Applications 40 — Medical Applica- tions 41 — Mass Screening of Natural Products will Utilize the Results of Engi- neered Proteins 43 — Growth Factors are a Promising Area for Protein Engi- neers 44 - The Marriage of Electronics and Biochemistry will Provide Many New Products 44 2 Analysis and Characterization of Proteins 47 Brigitte Wittmann-Liebold, Peter Jungblut 2.1 Introduction: How to Approach the Structure and Function of Pro- teins 47 By the Reductionistic Approach Individual Proteins are Selected for Investiga- tion 49 — By the Global Approach Single Proteins from Thousands of Polypep- tides in the Cell are Selected 50 2.2 The Various Preparative and Analytical Protein Purification Tech- niques 50 How to Consider an Appropriate Strategy for Protein Isolation 50 - Prefrac- tionation is Necessary for Reducing the Number of Proteins in a Complex Protein Mixture 53 — Strategies to Obtain Pure Proteins in a Few Steps 54 — Conventional Column Chromatography for Preparative Isolation of Pro- teins 54 - Gel Filtration is Based on Differences in Molecular Mass 54 - Ion Exchange Chromatography Allows the Isolation of Native Proteins 55 — Hy- drophobic Interaction Chromatography may Assist Isolation of Membrane Proteins 56 - Covalent Chromatography Binds Proteins to Supports for Selec- tive Separation 56 — Immobilized Metal Affinity Chromatography Uses Metal Chelate Formation 57 — Affinity Chromatography is Frequently Applied in Immunological Investigations 58 — HPLC-Separations and 2-DE-Polyacryl- amide Gel Electrophoresis Resolve Complex Protein Mixtures 60 — Electro- phoresis is Applied for Separating Highly Complex Protein Mixtures 60 — Electrophoresis in Combination with Blotting are Fast Means of Screening Pro- teins for Microsequencing and Immunostaining 65 — One Band in SDS Gel Electrophoresis Does Not Guarantee Purity of a Protein 67 — Desalting and Concentrating of Protein Fractions 69 — Spectroscopic Methods Can be Used for the Preliminary Quantitative Determination of Proteins 71 — Amino Acid Analysis is a First Step towards Characterizing a Protein 72 Contents IX 2.3 Investigation of Proteins and Protein Complexes 72 Identification of Subunits 73 - Extraction of Proteins from Hetero-Com- plexes 73 — End-Group Determinations Yield Information about Subunit Structures in Multi-Component Complexes 74 - Modern Mass Spectrometry Makes Possible Analysis of Proteins and Peptides 75 — Reduction and Alkyla- tion versus Treatment by Detergents 75 — Separation of Subunits 75 2.4 Strategies for Primary Structure Analysis of Proteins and Peptides. 76 Generation of Peptide Fragments 76 — Cyanogen Bromide Cleavage Releases Large Fragments for Sequence Alignment 76 — Mild Acid Treatment Gener- ates Peptides from Insoluble Proteins 77 - Enzymatic Protein Digestions Yield Suitable Peptides for Internal Sequence Analysis 77 - Assignment of Cysteine Residues and Cystine Bridges in Proteins 79 — Mass Spectrometry of Cysteine and Cystine Peptides 80 — Crosslinking of Neighboring Constituents 81 — Protein-Protein Crosslinking Yields Important Information about Distances in the Complex 82 — Investigation of the Crosslinked Proteins on the Amino Acid Level Yields a Valuable Fine Structure Analysis of the Complex 84 - Crosslinks between RNA (DNA) and Protein are More Difficult to Analyze than Protein-Protein Crosslinks 86 — Localization of Structural and Functional Domains 87 — Surface Peptides in Ribosomes 87 - Immunological Studies 88 — Anti-Protein Antibody Studies 88 - Synthetic Peptides are Useful for De- tecting Sequence-Specific Antigenic Sites 89 2.5 Protein Microsequence Analysis 90 The Chemistry of the Stepwise Edman Degradation Technique 91 — Advan- tages of the Automatic Microsequence Procedure 93 - Sample Preparation for Automated Sequence Analysis 94 - Manual Methods are Applied to Screen Many Peptide Samples Simultaneously 94 - Dansyl-Edman Degradation Yields Highly Sensitive Fluorescent Amino Acid Derivatives 95 — The Manual DABITC /PITC-Double Coupling Method is Used for Visual Detection of the Released Amino Acids in the Picomole Range 95 — Efforts to Develop Fluores- cent Isothiocyanates for a Sensitive Degradation Failed 96 — Manual Methods Allow the Simultaneous Degradation of Many Peptide Samples 96 — DNA- Sequencing of the Protein's Gene 97 - Animo Acid Sequencing is Necessary for Confirming the Deduced Gene Sequence 98 — Mass Spectrometric Meth- ods 98 — Mass Ion Determinations have Become Possible for Big Proteins Now 99 — Sequencing by Mass Spectrometry is a Complementary Technique to the Edman Degradation 100 — Determination of the C-Terminal Amino Acids is Difficult and of Limited Relevance 100 2.6 Conclusions 102 3 Structure Determination, Modeling and Site-directed Mutagenesis Studies 109 Ulrich Hahn, Udo Heinemann 3.1 Introduction 109 3.2 A Model System: Ribonuclease T1 110 Ribonuclease T1 is Small, Stable and Water-Soluble 112 — Single-Stranded RNA is Cleaved by Ribonuclease T1 Specifically after Guanine 112 X Contents 3.3 Methods of Structure Determination, Modeling and Mutagenesis . 114 The Three-Dimensional Structure of a Protein Molecule can be Determined with High Accuracy 115 — X-Ray Crystallography Yields High-Resolution Structures in the "Solid" State 117 — Nuclear Magnetic Resonance Spectros- copy Yields an Average Solution Conformation 122 - Databases Archive Structural Information 125 - Modeling and Force-Field Calculations Can Help in the Planning and Understanding of Mutagenesis Experiments 127 — Re- combinant DNA Technology Permits the Creation of Protein Molecules at Will 128 - Foreign DNA Fragments can be Biologically Amplified in Bacteria such as Escherichia coli 134 - High-Level Expression Provides the Amounts of Proteins Required for Many Experiments 138 — Site-Directed Mutagenesis Changes Protein Sequences according to Prespecified Goals 138 3.4 The Structures of Natural and Mutated Ribonuclease T1 145 The Structure of Wild-Type Ribonuclease T1 has been Determined by X-Ray Crystallography 146 — Ribonuclease T1-Inhibitor Complexes Provide Informa- tion about Enzyme Function 147 — Single Amino Acid Mutations may have Drastic Effect on Protein Function and Conformation 155 - NMR Spectros- copy Shows Protein Structure and Flexibility 156 3.5 Catalysis and Specificity 157 Active Site Mutations Help Us to Unterstand the Mechanism of RNA Hydroly- sis 158 - The Prediction of Protein Variants with Altered Substrate Specificity Remains a Problem 159 3.6 Folding Pathways 160 Ribonuclease T1 may Unfold and Refold with Complete Restoration of Activ- ity 160 — Cis-trans Isomerization of two Prolines is the Rate-Limiting Step in Ribonuclease T1 Folding 160 3.7 Protein Stability 161 The Stability of Ribonuclease T1 and Other