Abstract

The study addresses the critical challenge of characterizing protein-protein interactions (PPIs) involving membrane-associated enzymes, which is essential for understanding cellular signaling and metabolism. Specifically, this work investigates the application of Surface Plasmon Resonance (SPR) biosensing to analyze the interactions of Cytochrome P450 side-chain cleavage enzyme (P450scc or CYP11A1), a key mitochondrial enzyme in steroidogenesis. The primary objective was to establish a robust SPR-based methodology capable of measuring PPIs both in a conventional aqueous buffer and within a more physiologically relevant environment: a supported Bilayer Lipid Membrane (BLM). The methods involved immobilizing one interaction partner and monitoring the binding kinetics of the other, first in solution and then reconstituted into a BLM on the sensor surface. Key findings demonstrate that the SPR biosensor can successfully quantify the binding affinity and kinetics of P450scc with its redox partners, revealing significant differences in interaction parameters when the enzyme is embedded in the BLM compared to an aqueous environment. This highlights the crucial role of the lipid membrane in modulating the functional interactions of membrane proteins. The developed approach offers a powerful, label-free tool for the quantitative analysis of membrane protein complexes, providing deeper insights into their structure-function relationships and supporting drug discovery efforts targeting these vital cellular components.