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.

项目摘要 没有可靠的方法可以高精度稳定蛋白质。当前可用的方法具有 观察到突变对蛋白质稳定性的影响和预测影响之间的标准误差，范围为1至3 kcal/mol。鉴于蛋白质稳定性对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，开发可靠的方法来稳定蛋白质 高精度至关重要。为了以当前知识来解决这一辩护，我们已经开发了EMCAST（经验） C-Alpha稳定性工具）并表明它可以使小三个螺旋束的稳定性加倍，UBA（1）， 有四个突变。对于包含八个单，双重，三重和四倍的突变体变体 这四个突变的组合，预测和观察到的稳定性之间的平均误差为0.13 KCAL/MOL，对预测稳定突变的现有方法有了极大的改进。 emcast依靠两个 重要创新：1）使用源自Alpha Carbon（C）数据库的经验潜能 从2018年发行中提取的所有可能的四个救助序列的二面角偏好 蛋白质数据库和2）选择表面暴露的位点，以引入稳定突变。在 拟议的工作，我们将证明EMCAST可以成为稳定广泛蛋白质的有效工具 折叠，可以用来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Web服务，以便蛋白质生物化学社区 可以轻松访问并使用此有价值的工具。 我们将在以下目的的背景下实现这些目标： •在AIM 1中，纯和域的合理稳定，我们将证明Emcast可以稳定 具有高精度的另外四个螺旋结构域的集合，也可以应用于稳定 表域。这些蛋白质的预测稳定范围为2.5至6 kcal/mol。 •在AIM 2中，混合/域和大褶皱的稳定，我们应用EMCAST稳定集合 在包括-helix和表结构的四个复杂褶皱中，序列长度为 270个氨基酸。对于选定的蛋白质，Emcast预测稳定为3至5 kcal/mol。 •在AIM 3中，调节循环动力学并调整构象开关的位置，我们 将表明EMCAST可以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。