# Protein-induced membrane asymmetry modulates OMP folding kinetics and stability

## TL;DR

This study shows how proteins can influence membrane asymmetry, which in turn affects how proteins fold and remain stable in cell membranes.

## Contribution

A novel method using OmpA variants to create asymmetric proteoliposomes and study the interplay between protein and membrane dipoles.

## Key findings

- Matching the charge of the folding OmpA to the membrane dipole improves folding kinetics and stability.
- Protein-induced membrane dipoles significantly influence OMP folding behavior.
- Loop charge in membrane proteins may be used by cells to adapt membrane environments.

## Abstract

Biological membranes are asymmetric structures, with asymmetry arising from differences in lipid identity in each leaflet of the bilayer, as well as non-uniform distribution of lipids and small molecules in the membrane. Proteins can also induce and modulate membrane asymmetry based on their shape, sequence and interactions with lipids. How membrane asymmetry affects macromolecular behaviour is poorly understood because of the complexity of natural membrane systems, and difficulties in creating relevant asymmetric bilayer systems in vitro. Here, we present a method exploiting the efficient, unidirectional folding of the transmembrane β-barrel outer membrane protein, OmpA, to create asymmetric proteoliposomes with protein-induced dipoles of known direction (arising from sequence variation engineered into the OmpA loops). We then characterise the folding kinetics and stability of different OmpA variants into these proteoliposomes. We find that both the primary sequence of the folding OmpA and the dipole of the membrane into which folding occurs play an important role for modulating the rate of folding. Critically, we find that by complementarily matching the charge on the folding protein to the membrane dipole it is possible to enhance both the folding kinetics and the stability of the folded OmpA. The results hint at how cells might exploit loop charge in membrane-embedded proteins to manipulate membrane environments for adaptation and survival.

Complementary charge matching between a protein-induced membrane dipole and a folding OMP leads to optimal folding kinetics and protein stability.