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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

基本信息

批准号：
10709645
负责人：
BRUCE E BOWLER
金额：
$ 29.32万
依托单位：
UNIVERSITY OF MONTANA
依托单位国家：
美国
项目类别：
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

项目摘要

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.

项目摘要目前还没有可靠的方法可以高精度地稳定蛋白质。目前可用的方法有突变对蛋白质稳定性影响的观察结果和预测结果之间的标准误差范围为1到3 卡尔/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是至关重要的。为了解决当前知识的这一不足，我们开发了EmCAST(经验主义 C-Alpha稳定性工具)，并且已经表明它可以使小的三螺旋束UBA(1)的稳定性加倍，有四个突变。一组包含8个单、双、三和四个突变变体的将这四个突变组合在一起，预测的稳定性和观测的稳定性之间的平均误差为0.13 KCAL/摩尔，比现有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碳数据库(C)获得的经验势从2018年版本中提取的所有可能的四个残基序列的二面角偏好蛋白质数据库和2)选择表面暴露的位置以引入稳定突变。在在拟议的工作中，我们将证明EmCAST可以成为稳定广泛蛋白质的有效工具它可以用来调节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随时访问和使用这一有价值的工具。我们将在以下目标的框架内实现这些目标： ·在目标1，纯和结构域的理性稳定中，我们将证明EmCAST可以稳定高精度的四个附加螺旋域的集合，并且它也可以应用于稳定 -Sheet域的。预计这些蛋白质的稳定度在2.5到6千卡/摩尔之间。 ·在目标2，混合/结构域和大折叠的稳定化中，我们应用EMCAST来稳定一个集合四个同时包括-螺旋和-折叠结构的更复杂的折叠，其序列长度高达 270个氨基酸。EmCAST预测所选蛋白质的稳定化程度为3至5千卡/摩尔。 ·在目标3，调节环动力学和调整构象开关的位置，我们将表明EmCAST可以应用于蛋白质交替构象的差异化稳定，允许调节蛋白质功能。