Investigation of lipid/protein interactions in trifluoroethanol-water mixtures proposes the strategy for the refolding of helical transmembrane domains

Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol-water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol-water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains-transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.

Automated summary

Membrane proteins are essential in molecular biology, but studying their structures requires finding suitable membranes and refolding protocols. Isotropic bicelles are commonly used as membrane mimetics, but the refolding process is still not well understood. To find the best refolding methods for helical membrane proteins, we studied the interactions between proteins, lipids, and detergents in trifluoroethanol-water mixtures. Using NMR spectroscopy and light scattering, we determined key states of the compounds and proposed optimal protocols. These protocols were tested on various membrane domains with successful results.