S. Veit, L.C. Paweletz, S.S.-R. Bohr, A.K. Menon, N.S. Hatzakis and T. Günther Pomorski (2022).
Single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy.
ACS Applied Materials & Interfaces 14(26): 29659–29667
doi: 10.1021/acsami.2c07454
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.