Correlating membrane‐protein dynamics with function: Integrating bioinformatics, molecular dynamics, and single‐molecule <scp>FRET</scp>

We present a strategy that deploys structural bioinformatics, molecular simulation, and single-molecule Förster Resonance Energy Transfer (FRET) microscopy for observing the ligand-dependent conformational dynamics of integral membrane proteins in situ. We focus on representative members of the small monotopic phosphoglycosyl transferase (SmPGT) superfamily, which catalyze the transfer of a phosphosugar from a soluble nucleotide-sugar donor to a membrane-embedded polyprenol phosphate acceptor in the initiating step of glycoconjugate biosynthesis in prokaryotes. Substrate-specific structural features were identified across the superfamily and correlated with ligand-dependent conformational dynamics in all-atom simulations. To experimentally validate the role of this motion in ligand binding, we developed a platform to monitor intramolecular protein dynamics in a native-like lipid environment. The presented approach incorporates selective cysteine protein labeling and non-canonical amino acid mutagenesis with bicyclononyne-tetrazine click chemistry to assemble dual-labeled variants of PglC, the initiating enzyme of the N-linked protein glycosylation pathway from the Campylobacter genus. The modified proteins are solubilized in styrene-maleic acid liponanoparticles (SMALPs), which provide a model membrane environment. The conformational changes of PglC upon inhibitor binding correlate with inhibitor potency. The single-molecule FRET-SMALP strategy can be adapted to investigate protein dynamics across the superfamily of SmPGTs with different substrate selectivity, where structure prediction and molecular dynamics support significant conformational changes upon ligand binding.