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

膜转运蛋白是当前和未来药物的主要靶点，因为它们参与了许多导致人类疾病、细菌致病性、耐药性和其他医学现象的细胞过程。虽然转运蛋白的鉴定和结构分析已经取得了很大的进展，但仍然迫切需要更好地了解转运机制，以便开发新的治疗方法，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、能量使用和结构变化，促进对转运蛋白机制理解的下一个突破。虽然近年来在这方面取得了进展，主要是通过结合光学和电生理方法对离子通道的研究，但对于活性膜转运体的研究尚未取得类似的进展，因为活性膜转运体不适合电生理。本提案的目的是利用荧光传感器结合蛋白质和脂质体标记物，开发监测单个atp结合盒转运体（ABC转运体）的运输和能量转换过程的检测方法。这些新颖的实验工具将提供对初始转运速率、功能动力学（开/关周期）或单个转运体静态异质性的直接访问。本项目的主要目标是：目标1)开发和表征不同ABC转运体的荧光传感器，以监测底物易位，ATP水解和能量偶联。目的2)建立ABC转运蛋白的单转运蛋白记录，以观察随时间的功能动态或单个转运蛋白之间的静态差异。重要的是，通过同时进行脂质体大小表征，可以准确地量化初始转运速率。目的3)用开发的分析方法解决机制问题。详细地，我们将研究体和单转运体实验之间的差异，并表征催化NBD突变和不同底物对能量耦合效率的影响。

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.