Non-canonical amino acid mutagenesis in the engineering of insulin biophysics

胰岛素生物物理学工程中的非典型氨基酸诱变

• 批准号: 9803527
• 负责人: DAVID A TIRRELL
• 金额: $ 31.07万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9803527
• 关键词: Amino Acid Substitution; Amino Acids; Amino Acyl-tRNA Synthetases; Behavior; Binding; Biological Assay; Biophysics; Characteristics; Charge; Chemicals; Cold Chains; Computer Analysis; Computer software; Computing Methodologies; Diabetes Mellitus; Diabetic mouse; Dissociation; Engineering; Escherichia coli; Exhibits; FDA approved; Glucose; Human; Hydroxylation; Hydroxyproline; Injections; Insulin; Insulin, Aspart, Human; Insulin, Lispro, Human; Length; Life; Maintenance; Maps; Metabolic Diseases; Methods; Modeling; Modification; Molecular; Molecular Conformation; Mutagenesis; Pancreas; Periodicity; Pharmaceutical Preparations; Pharmacologic Substance; Play; Positioning Attribute; Preparation; Proline; Property; Protein Engineering; Proteins; Recombinant Proteins; Recombinants; Role; Savings; Structure; Therapeutic; Time; Variant; Work; absorption; analog; base; biophysical analysis; biophysical properties; cold temperature; design; diabetic patient; experimental analysis; flexibility; interest; mouse model; novel strategies; peptide drug; proline-tRNA; protein structure function; response; screening; subcutaneous; therapeutic protein

PROJECT SUMMARY/ABSTRACT We propose to explore the use of proline analogs to engineer and probe the biophysical behavior of insulin. Insulin contains a single proline residue – at position 28 of the B-chain – which is known to play critical roles in controlling the rates of onset of action and fibrillation of pharmaceutical preparations of the protein. Replacement of proline through conventional mutagenesis has led to FDA-approved rapid- acting insulins, but destabilizes the protein with respect to fibrillation. Conventional mutagenesis suffers from a fundamental limitation when applied to proline; any amino acid change converts the conformationally restricted cyclic proline residue to a more flexible acyclic one. We have recently found that replacement of the proline residue at position 28 of the insulin B-chain by (4S)-hydroxyproline – through non-canonical amino acid mutagenesis – yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. This approach allows one to alter critical molecular interactions around position B28 without sacrificing the unique conformational properties of proline. This proposal seeks to expand the known ‘proline chemical space’ that can be accessed in the bacterial expression of recombinant insulin. We will accomplish this objective by assessing the translational activity of a carefully chosen set of proline analogs in E. coli, by creating new prolyl-tRNA synthetases to activate the analogs of interest, and by analyzing the biophysical behavior of the resulting insulin variants by experimental and computational means. The proposed work will provide new forms of insulin with altered biophysical properties, expand the toolkit for engineering protein structure and function, and enhance our understanding of protein association and dynamics.

项目总结/摘要 我们建议探索使用脯氨酸类似物来工程化和探测生物物理行为， 胰岛素胰岛素在B链的28位含有一个脯氨酸残基， 在控制药物制剂的起效和原纤化速率方面的关键作用 蛋白质。通过常规诱变取代脯氨酸已经导致FDA批准的快速- 作用胰岛素，但使蛋白质在原纤化方面不稳定。传统的诱变方法 从一个基本的限制时，适用于脯氨酸;任何氨基酸的变化转化为 构象受限的环状脯氨酸残基转变为更灵活的非环状脯氨酸残基。我们最近发现 胰岛素B链28位的脯氨酸残基被（4S）-羟脯氨酸- 通过非规范氨基酸诱变-产生一种活性形式的胰岛素， 与野生型蛋白相比，其纤维化更快，并且纤维化更慢。这种方法允许改变关键的 B28位周围的分子相互作用，而不牺牲 脯氨酸。该提案旨在扩大已知的“脯氨酸化学空间”， 重组胰岛素的细菌表达。我们将通过评估 翻译活性的一组精心挑选的脯氨酸类似物在E。大肠杆菌，通过创造新的脯氨酰-tRNA 合成酶来激活感兴趣的类似物，并通过分析所得的类似物的生物物理行为， 通过实验和计算手段研究胰岛素变体。拟议的工作将提供新的形式， 改变生物物理特性的胰岛素，扩大了工程蛋白质结构的工具包， 功能，并增强我们对蛋白质缔合和动力学的理解。