Effects of Gly Residue and Cholesterol on the GXXXG-Mediated Parallel Association of Transmembrane Helices: A Single-Pair FRET Study.

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) helices (AALALAA AGLALGA AALALAA) 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.