No abstract

Small residue-mediated interhelical packing is ubiquitous in helical membrane proteins: however, the lipid dependence of its stability remains unclear. We previously demonstrated that the introduction of a GXXXG sequence in the middle of de novo-designed (AALALAA) 3 helices (AALALAA AGLALGA AALA-LAA) facilitated their dimerization, which was abolished by cholesterol. Here single-pair FRET measurements revealed that a longer GXXXGXXXG segment (AALALAA A GLALGA AAGALAA) promoted helix dimerization in POPC/cholesterol bilayers, but not without cholesterol. The predicted dimer structures and degrees of helix packing suggested that helix dimers with small (~10°) and large (~55°) crossing angles were only stabilized in POPC and POPC/cholesterol membranes, respectively. A steric hindrance in the dimer interface and the large flexibility of helices prevented the formation of stable dimers. Therefore, amino acid sequences and lipid compositions distinctively constrain stable dimer structures in membranes.

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.