Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors

Cells contain densely packed macromolecules that influence biochemical processes. The cytoplasm is known to be crowded, but membrane interfaces also exhibit similar conditions. Prior research has shown that proteins and nucleic acids occupy up to 30% of cellular volume. This crowding affects protein diffusion, conformation, and reaction kinetics. However, measuring membrane crowding remains a challenge. Existing methods struggle to quantify steric effects at lipid bilayers. This gap motivated the development of new tools to probe membrane environments. No prior work had resolved how membrane-bound crowders affect protein conformation. The need for non-invasive sensors became clear.

The goal was to create a genetically-encoded sensor for membrane crowding. The researchers aimed to measure how steric pressure alters protein conformation. They focused on membrane interfaces, where crowding is poorly understood. The study sought to establish procedures for membrane reconstitution. The team wanted to test sensor behavior in synthetic and native membranes. They also aimed to determine if molecular weight alone explains crowding effects. The motivation stemmed from the lack of tools to study membrane dynamics. This work could advance understanding of proteostasis in membranes.

The team designed two sensor variants with fluorescent proteins and membrane anchors. Each sensor differed in its flexible linker domain. In vitro characterization was performed to assess sensor behavior. Membrane reconstitution protocols were developed for experimental use. Synthetic and protein-based crowders were tethered to membranes. Fluorescence changes were measured to detect conformational shifts. Förster resonance energy transfer was used to quantify these changes. The sensors were tested in Escherichia coli inner membrane vesicles.

The sensors showed increased Förster resonance energy transfer under steric pressure. Membrane-tethered crowders induced conformational changes in the sensors. The effect was not strongly correlated with the molecular weight of crowders. Shape and charge of crowders likely influenced the observed dynamics. The sensors functioned in both synthetic and native membrane environments. Results suggest that quinary interactions contribute to crowding effects. The in vitro findings were validated in E. coli membrane vesicles. These results support the utility of the sensors for studying membrane crowding.

The authors propose that the sensors can probe membrane crowding in native environments. The findings suggest that factors beyond molecular weight affect crowding dynamics. The sensors offer a new method for studying membrane proteostasis. The team validated the sensors in both synthetic and physiological settings. The results support the use of Förster resonance energy transfer as a readout. The study highlights the importance of shape and charge in crowding effects. The sensors may help explore interfacial dynamics in complex membranes. These tools add to existing methods for membrane research.