Site-specifically mutated insulins have enormous potential value both ad hormones with improved physical, chemical and biological properties for the treatment of diabetes mellitus and as subjects for the study of insulin structure-function relationships. The Novo Research Institute (Denmark) had made available to this project large amounts (100 mg to >1 gm) of insulin mutants which vary widely in physical-chemical properties and in biological potency. Objectives: The long-term objective of this project is to further our knowledge of structure-function interrelationships for human insulin and proinsulin. The specific objectives are to answer the following questions: (a) How different are the solution structures of biologically active monomeric insulin and aggregated forms of metal-free native human insulin? (b) Is there a structure-function correlation between monomeric insulin mutants of widely varying biological activity? (c) Is there a significant difference in conformation between biologically active (monomeric) insulin and biologically inactive proinsulin? (d) How does protein concentration, ionic strength, pH and mutation at specific loci alter the aggregation and fibrillation behavior of metal-free insulin? (e) What hexamer conformation states are induced in solution by the lyotropic anions (SCN- ,l-,Br-, Cl-) and by phenol? (f) How are the energetics of the phenol and lyotropic anion-induced conformational transitions influenced by site- specific mutation? Relevance: The project is important to three areas of insulin therapy: (1) Understanding the relationship between insulin structure and the recognition of insulin by receptors; information critical to the rational design of improved insulins via genetic and chemical synthesis. (2) Understanding the effects of pH, ionic strength and site-specific mutation on the aggregation and fibrillation of insulin; information that is important for the improved design of insulin formulations for the treatment of insulin-dependent diabetics. (Insulin fibrillation is a serious obstacle to the development of a safe, implantable infusion pump for the delivery of insulin). (3) Understanding the dynamics of the lyotropic anion and phenol-induced conformation changes in the insulin hexamer; information that will assist efforts to design improved slow release formulations. Methods: NMR investigations of signatures of three-dimensional structure for various monomeric insulin species will use standard one- and two- dimensional methods (selective irradiation, magnetization transfer, multiple quantum filters, homonuclear 1H and heteronuclear 1H-15N multiple quantum correlation spectroscopy). 1H FT NMR, stopped-flow rapid-mixing kinetics and Doppler shift (dynamic photon correlation) light scattering spectroscopy will be used to study insulin aggregation and fibrillation and conformational transitions in the insulin hexamer.
