Table of Contents

• Abstract
• Acknowledgements
• Chapter 1 Discovering insulin
• 1.1 Introduction
• 1.2 Earliest treatment of Diabetes Mellitus
• 1.3 Towards reliable pancreas therapy
• 1.4 The insulin molecule
• 1.4.1 Finding the primary structure
• 1.4.2 The three-dimensional structure of insulin
• 1.4.3 Biosynthesis of insulin
• 1.4.4 Insulin function
• 1.5 Research into modern treatment of Diabetes Mellitus with insulin
• 1.6 Classification of Diabetes Mellitus
• 1.7 Studying the three-dimensional structure of insulin
• Chapter 2 Protein crystallographic methods
• 2.1 Introduction to protein crystallography
• 2.2 Crystallisation
• 2.3 Data collection and processing
• 2.4 Structure Determination
• 2.4.1 Isomorphous replacement
• 2.4.2 Molecular replacement
• The rotation function
• The translation function
• More about AMoRe
• 2.5 Refinement of protein structures
• 2.6 Maximum entropy and maximum likelihood
• 2.6.1 The concept of entropy
• 2.6.2 Maximum entropy
• Example I
• 2.6.3 Relative entropy
• Example II
• The minimum entropy principle
• 2.6.4 Bayes' theorem and likelihood
• Example III
• 2.6.5 Entropy and likelihood in protein crystallography
• Bricogne's Multisolution Tree
• REFMAC
• 2.7 Protein structure validation
• 2.7.1 R-factors
• 2.7.2 Estimates of coordinate precision
• Luzzati plot and Cruickshank's s_d
• Luzzati, Read and REFMAC: s_A
• 2.7.3 Stereochemistry and residue environment checks
• Ramachandran plot
• SQUID
• PROCHECK
• WHAT-IF and other geometry checking programs
• Folding profile methods
• 2.7.4 Brookhaven Protein Data Bank
• Chapter 3 Crystallographic studies of seven modified insulins
• 3.1 Introduction to modified insulins
• 3.1.1 B13 Glu®Gln human insulin
• 3.1.2 B9 Ser®Asp/B27 Thr®Glu human insulin
• 3.1.3 A21 Asn®Gly/B9 Ser®Glu/B10 His®Glu human insulin
• 3.1.4 Co²⁺ human insulin
• 3.1.5 B9 Ser®His human insulin
• 3.1.6 B8 Gly®Ser/B13 Glu®Gln/B30 Thr-NH₂ human insulin
• 3.1.7 A4 Glu®Gln/B25 Phe®Tyr/des B30 single chain human insulin
• 3.2 Crystallisation of the modified insulins
• 3.3 Data collection and processing
• 3.4 Structure Determination
• 3.4.1 Molecular replacement
• B9 Ser®Asp/B27 Thr®Glu human insulin
• A21 Asn®Gly/B9 Ser®Glu/B10 His®Glu human insulin
• A4 Glu®Gln, B25 Phe®Tyr, des B30 single chain human insulin
• 3.4.2 More self-rotations
• B13 Glu®Gln human insulin
• Zn²⁺®Co²⁺ human insulin
• B9 Ser®His human insulin, rhombohedral form
• B9 Ser®His human insulin, monoclinic form
• B8 Gly®Ser, B13 Glu®Gln, B30 Thr-NH₂ human insulin
• 3.5 Refinement
• 3.5.1 B13 Glu®Gln human insulin
• 3.5.2 Zn²⁺®Co²⁺ human insulin
• 3.5.3 B8 Gly®Ser, B13 Glu®Gln, B30 Thr-NH₂ human insulin
• 3.5.4 B9 Ser®His human insulin
• Monoclinic crystal form
• Rhombohedral crystal form
• 3.5.5 B9 Ser®Asp, B27 Thr®Glu human insulin
• 3.5.6 A21 Asn®Gly, B9 Ser®Glu, B10 His®Glu human insulin
• 3.5.7 A4 Glu®Gln, B25 Phe®Tyr, des B30 single chain human insulin
• 3.6 Description of the structure models
• 3.6.1 B13 Glu®Gln human insulin
• 3.6.2 B9 Ser®His human insulin
• Monoclinic crystal form
• Rhombohedral crystal form
• 3.6.3 B8 Gly®Ser, B13 Glu®Gln, B30 Thr-NH₂ human insulin
• 3.6.4 Zn²⁺®Co²⁺ human insulin
• 3.6.5 B9 Ser®Asp, B27 Thr®Glu human insulin
• 3.6.6 A21 Asn®Gly, B9 Ser®Glu, B10 His®Glu human insulin
• 3.6.7 A4 Glu®Gln, B25 Phe®Tyr, des B30 single chain human insulin
• Chapter 4 Insulin conformation
• 4.1 Introduction
• 4.2 A different hexamer, T3R3^f
• 4.3 The T ® R transition in modified insulin
• 4.4 Six glutamines in a single and a triple mutant
• 4.5 Summary
• Chapter 5 Aggregation of insulin
• 5.1 Introduction
• 5.2 Insulin dimerisation
• 5.2.1 Dimer stability
• 5.3 Insulin hexamerisation
• 5.3.1 Hexamer stability
• 5.4 Packing of insulin in crystals
• 5.5 Summary
• Chapter 6 Metal binding in insulin
• 6.1 Introduction
• 6.2 The role of zinc in insulin biosynthesis
• 6.2.1 Charges
• 6.3 The influence of phenol
• 6.4 Coordination of the metal
• 6.5 Other metals in other places
• 6.6 Summary
• Chapter 7 Conclusions and future work
• 7.1 Conclusions
• 7.2 Future work
• Appendix A Lagrange multipliers
• A.1 A general introduction to Lagrange multipliers
• A.2 Lagrange multipliers in entropy maximisation
• A.3 Lagrange multipliers in crystallography
• Appendix B Laboratory safety and reproduction
• B.1 Introduction
• B.2 Teratogenicity
• B.3 Biological hazards
• B.4 Chemical hazards
• B.4.1 The male parent
• B.4.2 The female parent
• B.5 Physical hazards
• B.5.1 Radiation
• B.5.2 Temperature and other possible stressors
• Bibliography