Long-range electrostatic interactions contribute to the target specificity and reactivity of thioredoxin family proteins

长程静电相互作用有助于硫氧还蛋白家族蛋白的靶标特异性和反应性

• 批准号: 251869040
• 负责人: Privatdozent Dr. Christopher Horst Lillig
• 金额: --
• 依托单位: Institut für Medizinische Biochemie und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251869040?language=en
• 关键词: Long; range; electrostatic; interactions; contribute

The spatio-temporal reduction and oxidation of protein thiols is an essential mechanism in signal transduction in all kingdoms of life. Thioredoxin (Trx) family proteins efficiently catalyse thiol-disulphide exchange reactions and the proteins are widely recognized for their importance in the operation of thiol switches. Trx family proteins have a broad and at the same time very distinct substrate specificity - a prerequisite for redox switching. Despite of multiple efforts, the basis for this specificity is still unclear. Our previous work suggests that thermodynamic parameters, such as the redox potential, do not determine specificity nor reactivity of the redoxins. Instead, the catalytic efficiency correlated strongly to a specific electrostatic field pattern of the redoxins [Chem. Sci. 6:7049-7058, 2015]. Efficient reaction rates rely on a sufficiently low activation energy barrier and a high frequency of effective collisions. Hence, we propose the following model: (1) The recognition of the proteins from a distance and their proper (pre-)orientation, dominated by long-range electrostatic interactions. (2) Attraction of the proteins towards each other. (3) Direct short-range molecular interactions, leading to the formation of the encounter complex. (4) The thiol-disulphide exchange reaction, subjected to a number of thermodynamic restrictions. (5) The dissociation of the complex. The primary aim of this project is to determine the importance of specific electrostatic interactions in the early phases (1-2) of the reaction. Most of all, we aim to establish the contributions of these forces to the specificity and overall rate constants of the reaction. The major focus of our research programme is the biochemical and biophysical characterisation of protein-protein interactions guided by computational analyses and predictions. As models, we propose to analyse the catalytic disulphide in E coli 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase by Grxs, the redox switch in human collapsin response mediator protein 2 2 (CRMP2) and, in cooperation with other members of the SPP 1710 consortium, other thiol switches of interest. We suggest to compute the electrostatic properties of the proteins using experimentally determined or modelled structures. Guided by these, we will analyse the interactions by enzyme kinetics, fluorescence quenching spectroscopy, surface plasmon resonance, and atomic force spectroscopy. We will determine the contribution of the association rates to the overall rate constants. Moreover, we aim to engineer specific interactions, for instance to turn E. coli Grx3 into an efficient reductant for PAPS reductase and to optimise the electron transfer between Grxs and the roGFP redox sensor. We have no doubt that the mechanisms that control the substrate specificity of Trx family proteins are key to the understanding of thiol switching.

蛋白质巯基的时空还原和氧化是所有生命王国中信号转导的重要机制。硫氧还蛋白（Trx）家族蛋白有效地催化巯基-二硫化物交换反应，并且该蛋白因其在巯基开关操作中的重要性而被广泛认识。Trx家族蛋白具有广泛的同时非常独特的底物特异性-这是氧化还原开关的先决条件。尽管多方努力，这种特异性的基础仍然不清楚。我们以前的工作表明，热力学参数，如氧化还原电位，不确定特异性，也不反应的氧化还原蛋白。相反，催化效率与氧化还原蛋白的特定静电场模式强烈相关[Chem. Sci. 6：7049-7058，2015]。有效的反应速率依赖于足够低的活化能势垒和高频率的有效碰撞。因此，我们提出了以下模型：（1）从远处识别蛋白质及其适当的（预）取向，由长程静电相互作用主导。(2)蛋白质相互吸引。(3)直接的短程分子相互作用，导致相遇复合物的形成。(4)硫醇-二硫化物交换反应受到许多热力学限制。(5)复合体的分裂。该项目的主要目的是确定反应早期阶段（1-2）特定静电相互作用的重要性。最重要的是，我们的目标是建立这些力量的贡献的特异性和整体反应速率常数。我们的研究计划的主要重点是通过计算分析和预测指导蛋白质-蛋白质相互作用的生物化学和生物物理特性。作为模型，我们建议通过Grxs分析大肠杆菌3 '-磷酸腺苷-5'-磷酸硫酸盐（PAPS）还原酶中的催化二硫化物，人胰蛋白酶反应介体蛋白2 2（CRMP 2）中的氧化还原开关，并与SPP 1710财团的其他成员合作，其他感兴趣的巯基开关。我们建议使用实验确定或建模的结构来计算蛋白质的静电性质。在此指导下，我们将通过酶动力学、荧光猝灭光谱、表面等离子体共振和原子力光谱来分析相互作用。我们将确定缔合速率对总速率常数的贡献。此外，我们的目标是设计特定的相互作用，例如将E。coli Grx 3转化为PAPS还原酶的有效还原剂，并优化Grxs和roGFP氧化还原传感器之间的电子转移。毫无疑问，控制Trx家族蛋白底物特异性的机制是理解巯基转换的关键。