Single-Molecule FRET Detection of GXXXG-Mediated Transmembrane Helix-Helix Interactions

Abstract

Helix-helix interactions in lipid bilayers are principal processes that determine the folding, oligomerization, and conformational change of helical transmembrane proteins. Not only the amino acid sequence of the protein but also the composition of surrounding lipids significantly affect the stability of the interaction. The GXXXG motif is frequently found at interaction interface of the transmembrane region, and proposed to mediate helix associations via hydrogen bonding between Cα-H donor and the backbone C=O acceptor. However, energetic/kinetic contributions of the motif have not been well characterized.In this study, we investigated the effect of a GXXXG-motif introduced into the center of the host transmembrane helix (AALALAA)3, examined by a single molecule FRET technique. The host helices are known to weakly self-associate in antiparallel orientations in POPC vesicles. In contrast, the GXXXG motif significantly stabilized a parallel association of the helices with lifetimes of subseconds. We also found that cholesterol suppressed the GXXXG-mediated parallel associations, demonstrating the importance of lipid environment on the helix-helix interaction