Binding orientation of weakly associating membrane peripheral proteins via membrane paramagnetic relaxation enhancement NMR.

Cell-membrane signaling and trafficking rely on proteins that associate with lipid bilayers through dynamic, low-affinity interactions. Defining how these proteins dock onto membrane surfaces is therefore essential for understanding their function. While Neutron Reflectometry (NR) combined with molecular dynamics (MD) simulations is frequently used, complementary approaches that do not require access to large-scale neutron facilities are needed. Here, we establish membrane Paramagnetic Relaxation Enhancement (mPRE) Nuclear Magnetic Resonance (NMR), combined with nanodiscs as membrane mimics and optimized acquisition strategies, as an accessible solution for extracting membrane-protein distance constraints even for weakly bound systems. Using the PI(4,5)P₂-binding ASAP1 Pleckstrin Homology (PH) domain as a model, we show that both conventional mPRE and a new dynamic-exchange mPRE (EX-mPRE) method reproduce the membrane orientation obtained by NR. In addition, we show that increasing PI(4,5)P₂ levels to mimic nanoscale membrane clustering broadened the orientational distribution of ASAP1 PH. EX-mPRE, which transfers PREs from transient bound states to the observable free state, further enables studies of temperature-sensitive or rapidly exchanging membrane interactions. Together, these results provide the formalism and establish mPRE and EX-mPRE NMR as a powerful alternative for resolving the membrane orientation of peripheral proteins and for probing how lipid composition affects their behavior.