Single Vesicle Fluorescence-Bleaching Assay for Multi-Parameter Analysis of Proteoliposomes by Total Internal Reflection Fluorescence Microscopy,ACS Applied Materials & Interfaces

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Single Vesicle Fluorescence-Bleaching Assay for Multi-Parameter Analysis of Proteoliposomes by Total Internal Reflection Fluorescence Microscopy
ACS Applied Materials & Interfaces ( IF 8.3 ) Pub Date : 2022-06-24 , DOI: 10.1021/acsami.2c07454
Sarina Veit ₁ , Laura Charlotte Paweletz ₁ , Søren S-R Bohr ₂ , Anant K Menon ₃ , Nikos S Hatzakis _{2,

4} , Thomas Günther Pomorski _{1,

5}

Affiliation

Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analysis of multiple parameters (physical size, tightness, unilamellarity, membrane protein content, and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.

中文翻译：

全内反射荧光显微镜单囊泡荧光漂白测定用于脂蛋白体多参数分析

将膜蛋白重建成模型膜是在化学定义的条件下进行功能分析的重要方法。用于整体平均测量的已建立的模型膜系统受到样品异质性以及对单个囊泡水平的脂质和蛋白质含量了解不足的限制，这限制了囊泡特性的定量分析并妨碍了它们与蛋白质活性的相关性。在这里，我们描述了一种基于全内反射荧光显微镜的通用漂白方案，该方案允许对用荧光标记的膜蛋白和脂质标记物制备的单个蛋白脂质体的多个参数（物理尺寸、紧密度、单层性、膜蛋白含量和方向）进行并行分析。该方法利用市售荧光团，包括常用的硝基苯并恶二唑染料，可用于推断多种类型的重构荧光标记膜蛋白的功能分子特征。

更新日期：2022-06-24

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