Interplay between Amino Acid Sequences and Lipid Compositions in the GXXXG-Mediated Parallel Self-Association of Transmembrane Helices as Revealed by Single-pair Fret

Abstract

Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We introduced GXXXG motifs in a pair of de-novo designed (AALALAA)3 helices incorporated into POPC liposomes with their topology controlled in a parallel fashion, and their self-association was monitored by single-pair FRET in real time. The introduction of a GXXXG sequence in the middle of the sequence (AALALAA AGLALGA AALALAA) facilitated the dimerization of helices, which was abolished by cholesterol. In contrast, the incorporation of a longer GXXXGXXXG segment (AALALAA AGLALGA AAGALAA) promoted the self-association of helices in POPC/cholesterol bilayers, but not without cholesterol. The inclusion of a longer motif (AALALGA AGLALGA AGLALAA) nullified the dimerization. Furthermore, the dimerization of GXXXG-introduced helices was found highly sensitive to amino acid sequences flanking the GXXXG motif and its position along the entire sequence. Underlining mechanisms will be discussed.