EmCAST: Stabilizing Proteins and Tuning Dynamics with High Precision and Accuracy

EmCAST：以高精度和准确度稳定蛋白质并调节动力学

• 批准号: 10566514
• 负责人: BRUCE E BOWLER
• 金额: $ 29.33万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-24 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10566514
• 关键词: Address; Affect; Algorithms; Amino Acids; Attenuated; Binding; Biological Assay; Biosensor; Carbon; Communities; Complex; Data; Databases; Development; Directed Molecular Evolution; Equilibrium; Goals; Kinetics; Knowledge; Length; Life; Link; Measures; Methods; Molecular Conformation; Mutation; Output; Pharmacologic Substance; Positioning Attribute; Probability; Protein Biochemistry; Protein Conformation; Protein Dynamics; Proteins; Publishing; Research; Research Project Grants; Site; Solvents; Specificity; Structure; Surface; Testing; Variant; Vertebral column; Work; base; conformer; immunogenicity; improved; innovation; mutant; preference; protein data bank; protein folding; protein function; protein structure; rational design; scaffold; success; therapeutic protein; tool; web server; web services

Project Summary There are no reliable methods to stabilize proteins with high accuracy. Currently available methods have standard errors between observed and predicted effects of mutations on protein stability that range from 1 to 3 kcal/mol. Given the importance of protein stability for biomedical applications such as the shelf-life and immunogenicity of protein-based pharmaceuticals, development of reliable methods to stabilize proteins with high accuracy is critical. To address this deficit in current knowledge, we have developed EmCAST (Empirical C-Alpha Stability Tool) and have shown that it can double the stability of a small three helix bundle, UBA(1), with four mutations. For a set of eight single, double, triple, and quadruple mutant variants that contain combinations of these four mutations, the average error between predicted and observed stability was 0.13 kcal/mol, a vast improvement over existing methods to predict stabilizing mutations. EmCAST relies on two important innovations: 1) use of an empirical potential derived from a database of the alpha carbon (C) dihedral angle preferences for all possible four-residue sequences extracted from the 2018 release of the Protein Data Bank and 2) selection of surface-exposed sites for introduction of stabilizing mutations. In the proposed work, we will demonstrate that EmCAST can be an effective tool to stabilize a broad range of protein folds and that it can be used to tune the position of protein conformational switches and hence control protein function. We will also release, maintain, and upgrade a web service so that the protein biochemistry community can readily access and use this valuable tool. We will accomplish these goals in the context of the following Aims: • In Aim 1, Rational Stabilization of Pure  and  Domains, we will show that EmCAST can stabilize a set of four additional helical domains with high accuracy and that it can also be applied to stabilization of -sheet domains. Predicted stabilizations for these proteins range from 2.5 to 6 kcal/mol. • In Aim 2, Stabilization of Mixed / Domains and Large Folds, we apply EmCAST to stabilize a set of four more complex folds that include both -helix and -sheet structure with sequence lengths up to 270 amino acids. EmCAST predicts stabilizations of 3 to 5 kcal/mol for the selected proteins. • In Aim 3, Regulating Loop Dynamics and Tuning the Position of Conformational Switches, we will show that EmCAST can be applied to differential stabilization of alternate conformers of proteins, allowing for tuning of protein function.

项目概要 没有可靠的方法来高精度稳定蛋白质。目前可用的方法有 观察到的和预测的突变对蛋白质稳定性的影响之间的标准误差，范围为 1 到 3 千卡/摩尔。鉴于蛋白质稳定性对于生物医学应用的重要性，例如保质期和 基于蛋白质的药物的免疫原性，开发稳定蛋白质的可靠方法 高精度至关重要。为了解决当前知识的这一缺陷，我们开发了 EmCAST（经验 C-Alpha 稳定性工具），并表明它可以使小型三螺旋束 UBA(1) 的稳定性加倍， 有四个突变。对于一组八个单、双、三和四突变体变体，其中包含 这四种突变的组合，预测稳定性和观察到的稳定性之间的平均误差为 0.13 kcal/mol，比现有的预测稳定突变的方法有了巨大的改进。 EmCAST 依赖于两个 重要创新：1) 使用从 α 碳 (C) 数据库得出的经验潜力 从 2018 年版本中提取的所有可能的四残基序列的二面角偏好 蛋白质数据库和 2) 选择表面暴露位点以引入稳定突变。在 拟议的工作中，我们将证明 EmCAST 可以成为稳定多种蛋白质的有效工具 折叠，它可用于调整蛋白质构象开关的位置，从而控制蛋白质 功能。我们还将发布、维护和升级网络服务，以便蛋白质生化社区 可以轻松访问和使用这个有价值的工具。 我们将在以下目标的背景下实现这些目标： • 在目标 1，纯  和  域的合理稳定中，我们将证明 EmCAST 可以稳定 一组四个附加螺旋域，具有高精度，也可应用于稳定 -片域。这些蛋白质的预计稳定范围为 2.5 至 6 kcal/mol。 • 在目标 2，混合 α/β 结构域和大折叠的稳定中，我们应用 EmCAST 来稳定一组 四个更复杂的折叠，包括 α 螺旋和 β 片层结构，序列长度可达 270个氨基酸。 EmCAST 预测所选蛋白质的稳定性为 3 至 5 kcal/mol。 • 在目标 3 中，调节环路动力学并调整构象开关的位置，我们 将表明 EmCAST 可应用于蛋白质替代构象异构体的差异稳定， 允许调整蛋白质功能。