Backbone Nitrogen Substitution Probes the Role of Glycine Residues at GxxxG Interfaces in Transmembrane Helices

Association of transmembrane helices is central to the structure and function of membrane proteins. A common feature of these interfaces is the GxxxG motif, which allows for close contact between the protein backbones, resulting in the formation of a network of weak hydrogen bonds between C α –H donors and carbonyl acceptors on opposing helices (i.e., C α –H···O═C hydrogen bonds). Engineering these interfaces holds promise for the control of membrane protein activity, but few strategies exist for the substitution of the critical glycine residues, which lack mutable side chains. Herein, we replace glycine residues at the dimerization interface of single-pass transmembrane proteins (i.e., glycophorin A and BNIP3) with aza-glycine, which substitutes the α-carbon with nitrogen. We hypothesized that aza-glycine incorporation would enhance intermolecular association by introducing a stronger hydrogen-bond donor and increasing solvophobic interactions within the lipid bilayer. In contrast, FRET-based measurements of helix association showed that aza-glycine substitution was insufficient to increase dimerization affinity, which we attribute to the decreased helical propensity of aza-glycine. Our results demonstrate a synthetic strategy to probe the determinants of membrane-protein folding and inform the design of transmembrane peptidomimetics.