Work

Real-Time Binding Kinetics of Membrane Protein–Protein Interactions in a Membraneless Setting

Year: 2025

Type: article

Abstract: A ubiquitous problem in protein analytics and medical biotechnology is assessing the interaction of a membrane protein receptor with its cognate protein ligand. This task generally requires transferring the receptor from native membranes or other expression host systems into supported lipid bilayers, liposomes, or nanodiscs. Such a reintegration process necessitates multiple steps for protein solubilization, renaturing, and functional reconstitution. Here, we opportunistically show that biolayer interferometry (BLI) can be directly utilized to evaluate the pre-equilibrium binding kinetics of a membrane protein receptor with its protein ligand in a label-free and membraneless setting. We present real-time measurements probing the association and dissociation phases of these transient complexes, conducted at a high signal-to-noise ratio using free proteomicelles in solution. As a proof-of-concept, we employ a subset of synthetic membrane proteins equipped with a programmable antibody mimetic binder that targets a specific protein ligand. Proteomicelles containing these binder-equipped membrane proteins exhibit high-affinity interactions with ligands attached to the sensor surface. These determinations are further validated by closely related surface plasmon resonance (SPR) measurements of the binder-ligand and proteomicelle-ligand interactions. Finally, this approach is amenable to high-throughput data collection, and its conceptual formulation is potentially extendable to other membrane proteins.

Source: Analytical Chemistry

DOI: 10.1021/acs.analchem.5c05510