Quantitative Paramagnetic NMR-Based Analysis of Protein Orientational Dynamics on Membranes: Dissecting the...

Peripheral membrane proteins can adopt distinct orientations on the surfaces of lipid bilayers that are often short-lived and challenging to characterize by conventional experimental methods. Here we describe a robust approach for mapping protein orientational landscapes through quantitative interpretation of paramagnetic relaxation enhancement (PRE) data arising from membrane mimetics with spin-labeled lipids. Theoretical analysis, followed by experimental verification, reveals insights into the distinct properties of the PRE observables that are generally distorted in the case of stably membrane-anchored proteins. To suppress the artifacts, we demonstrate that undistorted Γ₂ values can be obtained via transient membrane anchoring, based on which a computational framework is established for deriving accurate orientational ensembles obeying Boltzmann statistics. Application of the approach to KRas4B, a classical peripheral membrane protein whose orientations are critical for its functions and drug design, reveals four distinct orientational states that are close but not identical to those reported previously. Similar orientations are also found for a truncated KRas4B without the hypervariable region (HVR) that can sample a broader range of orientations, suggesting a confinement role of the HVR geometrically prohibiting severe tilting. Comparison of the KRas4B Γ₂ rates measured using nanodiscs containing different types of anionic lipids reveals identical Γ₂ patterns for the G-domain but different ones for the HVR, indicating only the latter is able to selectively interact with anionic lipids.