Single-transporter biochemistry: real-time observation of substrate transport and energy conversion of primary-active membrane transporters

单转运蛋白生物化学：实时观察初级活性膜转运蛋白的底物转运和能量转换

• 批准号: 494983708
• 负责人: Professor Dr. Thorben Cordes
• 金额: --
• 依托单位: Abteilung Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/494983708?language=en
• 关键词: Single; transporter; biochemistry; real; time

Membrane transporters are prime targets for current and future pharmaceutical drugs due to their involvement in numerous cellular processes that cause human diseases, bacterial pathogenicity, drug resistance, and other medical phenomena. Whereas the identification and structural analysis of transporters has moved at great pace, there is still an urgent need to better understand transport mechanisms to enable the development of novel therapies, e.g., through the identification of inhibitors. Single-molecule-based approaches could facilitate a next breakthrough in mechanistic understanding of transporters via (simultaneous) real-time observation of transport, energy usage and structural changes. Although there have been recent advances into this direction, mainly in the investigations of ion channels by combining optical and electrophysiological methods, similar progress has not been made for active membrane transporters, which are not amenable to electrophysiology. The objectives of this proposal are to develop assays to monitor transport and energy conversion processes of single ATP-binding cassette transporters (ABC transporters) using fluorescent sensors in combination with protein and liposome markers. These novel experimental tools will provide direct access to initial transport rates, functional dynamics (on/off periods) or static heterogeneity of single transporters. The main goals of this project are:Aim 1) Development and characterization of fluorescent sensors for different ABC transporters to monitor substrate translocation, ATP hydrolysis and energy coupling. Aim 2) Establish single-transporter recordings for ABC transporters to observe functional dynamics over time or static differences between individual transporters. Importantly, the initial rates of transport will be accurately quantified via simultaneous liposome size characterization.Aim 3) Solve mechanistic questions with the developed assays. In detail, we will study the difference between bulk and single transporter experiments and characterize the impact of catalytic NBD mutations and different substrates on energy coupling efficiencies.

膜转运蛋白是当前和未来药物的主要靶点，因为它们参与了导致人类疾病、细菌致病、耐药性和其他医学现象的众多细胞过程。尽管转运蛋白的鉴定和结构分析进展很快，但仍迫切需要更好地了解转运机制，以便能够开发新的治疗方法，例如通过鉴定抑制剂。基于单分子的方法可以通过(同时)实时观察运输、能源使用和结构变化，促进对转运体的机械理解的下一次突破。尽管最近在这方面已经取得了一些进展，主要是通过光和电生理相结合的方法来研究离子通道，但对于不受电生理影响的活性膜转运体却没有取得类似的进展。这项建议的目标是开发监测单一的三磷酸腺苷结合盒转运体(ABC转运体)的运输和能量转换过程的分析方法，结合使用荧光传感器和蛋白质和脂质体标记。这些新颖的实验工具将提供对单个转运体的初始传输速率、功能动态(开/关期)或静态异质性的直接访问。本项目的主要目标是：1)开发和表征用于不同ABC转运蛋白的荧光传感器，以监测底物转运、ATP水解酶和能量耦合。目的2)建立ABC转运蛋白的单一转运体记录，以观察功能随时间的动态变化或单个转运体之间的静态差异。重要的是，最初的转运速率将通过同步的脂质体大小特征被准确地量化。目的3)用所开发的检测方法解决机制问题。具体来说，我们将研究散装和单一转运子实验之间的差异，并表征催化NBD突变和不同底物对能量耦合效率的影响。