Quantitative and mechanistic assessment of thiol switches by stopped-flow kinetic measurements

通过停流动力学测量对硫醇开关进行定量和机理评估

• 批准号: 249669453
• 负责人: Professor Dr. Marcel Deponte
• 金额: --
• 依托单位: Fachrichtung Biochemie
• 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 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/249669453?language=en
• 关键词: Quantitative; mechanistic; assessment; thiol; switches

Background:Several thiol switch candidates have been identified and characterized during the 1st funding period of the SPP 1710. Such characterizations are often based on redox mobility shift assays and protein-protein interaction studies. Quantitative kinetic and detailed mechanistic data on potential as well as established switches are usually difficult to obtain using such methods. Hence, the underlying rate constants for toggling thiol switches and the physiological relevance of detected redox species often remain unclear. Objectives:During the 2nd funding period of the SPP 1710, we will focus on the quantitative and mechanistic assessment of (candidate) thiol switches by stopped-flow kinetic measurements. We will therefore complement the portfolio of existing key methods within the priority programme by establishing a stopped-flow platform. This platform will be accessible to all SPP members who are interested in the determination of rate constants for (candidate) thiol switches and the elucidation of mechanisms for thiol switching and roGFP2-based redox sensing. For example, we will assess the reactivity of the cysteine residues of the Keap1-like artemisinin-resistance factor PfK13 from the malaria parasite Plasmodium falciparum and characterize the regulatory role of residues C143 & L109 for the Prx5-type model peroxiredoxin PfAOP. Other peroxiredoxins as well as glutaredoxins, roGFP2 probes and flavoenzymes will be studied in the frame of collaborations with SPP 1710 members. Findings from this project will improve our basic knowledge on thiol switches and redox probes and might also help to better understand redox signaling and artemisinin resistance of malaria parasites.

背景：在SPP 1710的第一个资助期内，已经确定并表征了几种硫醇开关候选物。这种表征通常基于氧化还原迁移率变动测定和蛋白质-蛋白质相互作用研究。定量动力学和详细的机制数据的潜力，以及建立开关通常很难获得使用这样的方法。因此，切换硫醇开关的基本速率常数和检测到的氧化还原物质的生理相关性通常仍不清楚。目标：在SPP 1710的第二个资助期内，我们将重点关注通过停流动力学测量对（候选）硫醇开关进行定量和机制评估。因此，我们将通过建立一个停止流动平台，补充优先方案内现有的关键方法组合。该平台将可供所有SPP成员访问，他们对（候选）硫醇开关的速率常数的确定以及硫醇开关和基于roGFP 2的氧化还原传感机制的阐明感兴趣。例如，我们将评估来自疟疾寄生虫恶性疟原虫的Keap 1样青蒿素抗性因子PfK 13的半胱氨酸残基的反应性，并表征残基C143和L109对Prx 5型模型过氧化物氧还蛋白PfAOP的调节作用。将在与SPP 1710成员合作的框架内研究其他过氧化物酶以及谷氧还蛋白、roGFP 2探针和黄素酶。该项目的发现将提高我们对巯基开关和氧化还原探针的基础知识，也可能有助于更好地了解疟疾寄生虫的氧化还原信号和青蒿素抗性。