Abstract
Protein behavior in lipid is poorly understood and inadequately represented in current computational models. Design and prediction abilities for bilayer-embedded molecular structures may be improved by characterizing membrane proteins' most frequent, favored structural features to glean both context-specific and general principles. We used protein design to proactively interrogate the sequence-structure relationship and stabilizing atomic details of two highly prevalent antiparallel transmembrane (TM) motifs with Small-X (6) -Small consensus sequences. A fragment-based data-mining and sequence statistical inference method including cross-evolutionary structure-aligned covariance enabled engineering of de novo multi-span TM protein assemblies by successfully encoding Gly-X6-Gly and Ala-X6-Ala building blocks. A highly stable glycine-based design's X-ray structure hosts Cα-H···O=C H-bonding alongside extensive backbone-directed van der Waals packing, idealizing features of this motif in Nature. Data-driven design navigates sequence space to directly inquiry upon how to encode and stabilize vital membrane protein structural elements, facilitating efficacious construction of lipid-embedded architectures of increasing complexity. SIGNIFICANCE: Membrane proteins comprised of α-helices pack together within lipid bilayers, establishing stabilities and architectures that brace function and guide evolution. De novo design was used to clarify the consensus sequences and molecular features encoding of one exceedingly common TM helix packing architecture (∼10% of those in membrane folds). Small-X (6) -Small residue patterns were proven to reliably drive minimal proteins into these antiparallel TM geometries, revealing glycine mainchain hydrogen bonding or via fully apolar interfaces can encode the motif. Optimizing steric packing was the most decisive feature amongst the synthetic proteins. New membrane-specific design methods and model molecules are validated in route to outlining this important sequence-structure relationship broadly impacting the membrane proteome and revealing generalized structure-energetic imperatives governing interactions in lipid.