Quantitative Membrane Paramagnetic Relaxation Enhancement (mPRE) Measurements on Lipid-Anchored Proteins: Insight...

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.