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

## TL;DR

This paper introduces a new method using biolayer interferometry to study membrane protein interactions in real time without membranes.

## Contribution

The novel use of biolayer interferometry for label-free, membraneless assessment of membrane protein–protein interactions is demonstrated.

## Key findings

- Biolayer interferometry enables real-time measurement of membrane protein binding kinetics in a membraneless environment.
- Proteomicelles with antibody mimetic binders show high-affinity interactions validated by surface plasmon resonance.
- The method is suitable for high-throughput data collection and potential extension to other membrane proteins.

## 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.