CHEMICAL AND STRUCTURAL SIGNATURES OF INSULIN FUNCTION

胰岛素功能的化学和结构特征

• 批准号: 3243134
• 负责人: MICHAEL F DUNN
• 金额: $ 9.76万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3243134
• 关键词: acidity /alkalinity; anions; bioenergetics; chemical aggregate; conformation; human tissue; insulin; insulin receptor; light scattering; monomer; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; peptide hormone; peptide hormone analog; proinsulin; protein structure function; site directed mutagenesis; solutions; stop flow technique

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.

位点特异性突变的胰岛素具有巨大的潜在值AD 具有改善物理，化学和生物学特性的激素 糖尿病的治疗和作为胰岛素研究的受试者 结构功能关系。 Novo Research Institute（丹麦） 已向该项目提供了大量（100毫克至> 1克） 胰岛素突变体在物理化学特性和 生物效力。 目标：该项目的长期目标是进一步 了解人类胰岛素和 Proinsulin。 具体目标是回答以下问题： （a）生物活性的溶液结构有多不同 单体胰岛素和无金属本地胰岛素的聚集形式？ （b）单体胰岛素之间是否存在结构功能相关 广泛变化的生物活性的突变体？ （c）有重要的 生物活性（单体）胰岛素之间构象的差异 和生物学上无活性的促素？ （d）蛋白质浓度如何 特定基因座的离子强度，pH和突变改变了聚集 无金属胰岛素的纤颤行为？ （e）什么己隔 通过溶解阴离子诱导构象状态（SCN- ，l-，br-，cl-）和苯酚？ （f）酚和苯酚的能量如何 由位点影响 - 具体突变？ 相关性：该项目对胰岛素治疗的三个领域很重要： （1）了解胰岛素结构与 通过受体识别胰岛素；对理性至关重要的信息 通过遗传和化学合成改善胰岛素的设计。 （2） 了解pH，离子强度和特定位点突变的影响 关于胰岛素的聚集和纤颤；就是 对于改善治疗的胰岛素制剂设计很重要 胰岛素依赖性糖尿病患者。 （胰岛素纤颤是严重的 开发安全，可植入的输液泵的障碍 胰岛素的递送）。 （3）理解溶解的动力学 阴离子和苯酚诱导的构象变化，胰岛素己酰胺的变化； 将有助于设计改进缓慢释放的努力的信息 配方。 方法：三维结构标志的NMR研究 对于各种单体胰岛素物种，将使用标准的一单和二 维方法（选择性照射，磁化转移， 多个量子过滤器，同核1H和异核1H-15N多重 量子相关光谱）。 1H ft NMR，停止流量快速混合 动力学和多普勒移位（动态光子相关）光散射 光谱法将用于研究胰岛素聚集和原纤维， 胰岛素六聚体中的构象过渡。