Abstract

The function of many lipid-anchored eukaryotic proteins critically depends on their specific orientations relative to the membranes, which can block key active centers or binding interfaces. A precise mapping of these orientational landscapes is therefore vital for elucidating/understanding protein function, yet it remains technically challenging. Although membrane paramagnetic relaxation enhancement (mPRE) NMR spectroscopy can, in principle, map membrane–protein interfaces, the apparent observables can be drastically distorted for irreversibly lipid-anchored proteins. Here, we overcome this limitation by developing a tunable lipidation strategy using synthetic alk-1-en-3-one reagents. We demonstrate that conjugating undec-1-en-3-one to target proteins creates an effective anchor that establishes the fast-exchange equilibrium required for accurate mPRE measurements, restoring the theoretical dependence between apparent Γ2 rates and interspin distances. We validated this approach with a ubiquitin-based model system and then applied it to quantify the orientational dynamics of the oncoproteins KRas4B and HRas. A comparative analysis of the orientational ensembles constructed with artifact-free mPRE data reveals that the negatively charged POPS influences KRas4B orientations indirectly by constraining its C-terminal polybasic region, reshaping the landscape in favor of signaling-compatible orientations. Furthermore, we find that HRas on the neutral POPC bilayers adopts an orientational distribution strikingly similar to KRas4B on anionic, but not neutral, membranes, suggesting a functionally essential orientational convergence. This work establishes a generalizable method for quantifying membrane orientations of lipidated proteins, providing new insights into Ras regulation and opening the door to studying many previously intractable lipid-anchored systems.