Interfacial structure and protein incorporation in sparsely tethered phospholipid membranes

Tethered bilayer lipid membranes (tBLMs) are a robust model system for studying the biophysics of cell membranes, including protein-lipid interactions and membrane dynamics. In this study we describe the structural properties of a novel tBLM platform based on self-assembled monolayers (SAMs) on gold presenting sparsely distributed linear tethers. The interfacial architecture of tBLMs built on two types of alkane tether arrangements, homogeneously distributed short tethers and nanoclustered long tethers, were resolved using neutron reflectometry (NR). A series of tBLM systems was prepared and structurally characterized, with variations in membrane phase (gel and fluid lipids), substrate attachment type (floating and tethered), and electrostatic properties (zwitterionic and negatively charged lipids). Furthermore, the versatility of the tBLM platform was demonstrated by incorporating transmembrane proteins, specifically the outer membrane protein F (OmpF), into the tethered bilayer. Quantitative analyses using NR and quartz crystal microbalance with dissipation monitoring (QCM-D) confirmed successful protein incorporation, with an estimated OmpF volume fraction ∼ 18 % within the tBLM. The tBLMs exhibited excellent stability and maintained structural integrity under continuous flow conditions during up to 16-hour NR experiments. Our results highlight the adaptability of this sparse tethering system for creating physiologically relevant membrane models, facilitating precise investigations of membrane-associated processes and protein interactions. The study establishes the potential of this platform for advancing biophysical research on cell membranes and membrane proteins, as well as developing biomimetic systems for analytical and screening applications.