Triangulating a Tightly Bound Lipid on a Membrane Protein by Paramagnetic Solid-State NMR

Nonannular lipids tightly associate with membrane proteins, influencing their structure and function. Here, we present a solid-state NMR (ssNMR)-based strategy to localize such lipids on protein surfaces with a high specificity. We previously identified a tightly bound glycophospholipid which copurifies with Anabaena Sensory Rhodopsin trimers and displays resolved NMR signals. By introducing paramagnetic labels at defined sites and measuring paramagnetic relaxation enhancements (PREs), we triangulate the lipid's position near a periplasmic intermonomer cleft. Docking calculations guided by PRE restraints further define a nonannular lipid binding site. Our approach provides a broadly applicable framework for mapping tightly bound lipids in membrane proteins under native-like conditions.