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

项目摘要/摘要 我们建议探索使用脯氨酸类似物来设计和探索生物物理行为 胰岛素。胰岛素在B链的第28位含有单一的脯氨酸残基，这是已知的作用 在控制药物制剂的起效和纤颤发生率中的关键作用 蛋白质。通过传统的诱变取代脯氨酸已经导致了FDA批准的快速- 作用胰岛素，但在纤颤方面破坏蛋白质的稳定。传统的诱变技术遭受了 当应用于Pro时，从根本限制；任何氨基酸的变化都会将 构象受限的环状脯氨酸残基转变为更灵活的无环残基。我们最近发现 将胰岛素B链第28位的脯氨酸残基替换为(4S)-羟脯氨酸- 通过非标准氨基酸突变-产生一种活性形式的胰岛素，它能更多地解离 与野生型蛋白质相比，它的纤化速度更快，速度更慢。此方法允许更改关键的 B28位附近的分子相互作用而不牺牲 脯氨酸。这项提议寻求扩大已知的可在 重组胰岛素的细菌表达。我们会通过评估 通过创建新的Pro-tRNA，在大肠杆菌中精心选择一组Pro类似物的翻译活性 合成酶激活感兴趣的类似物，并通过分析结果的生物物理行为 胰岛素变异的实验和计算方法。拟议的工作将提供新的形式 具有改变生物物理性质的胰岛素，扩展用于工程蛋白质结构和 功能，并增强我们对蛋白质关联和动力学的理解。

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.