Quantitative Kinetic Analysis of Protein-Phosphoinositide Binding by Optimized Liposome-Based Bio-Layer...

Phosphoinositide-binding domains recruit cytosolic proteins to specific membrane surfaces, and their binding specificity and kinetics are critical determinants of protein localization and signaling. However, most available assays report only equilibrium binding strength and do not resolve the underlying association and dissociation kinetics. Here, we developed an optimized liposome-based bio-layer interferometry (BLI) assay for specificity profiling and comparative kinetic analysis of protein-phosphoinositide interactions. Buffer optimization showed that 0.5% bovine serum albumin and 0.001% Tween-20 almost completely suppressed nonspecific protein adsorption to the biosensor surface while preserving the expected specific interactions between proteins and liposomes. Using representative PX and PH domain proteins and binding-altered mutants, we obtained phosphoinositide-binding profiles consistent with previous biochemical data. Concentration-dependent BLI analysis further resolved distinct kinetics contributing to affinity enhancement: for a PX-SnxA mutant series, stronger apparent binding was associated primarily with increased association rates, whereas for PH-AKT1 mutants, it was associated primarily with decreased dissociation rates. These results show that the platform can attribute differences in apparent affinity to specific changes in association and/or dissociation rates, providing a level of mechanistic resolution useful for both characterizing and engineering lipid-binding probes.