Biolayer Interferometry for Investigating Membrane Protein-Inhibitor Binding: TACAN Mutant and GsMTx4 As a Model...

Bio-Layer Interferometry (BLI) is an efficient biophysical technique that enables real-time, quantitative analysis of biomolecular interactions. Although commonly used for soluble proteins, their application to membrane proteins remains challenging due to hydrophobicity and detergent dependence. This study presents an optimized BLI protocol for characterizing interactions between detergent-solubilized membrane protein and peptide inhibitor. As a model system, the gating-modifier peptide Grammostola mechanotoxin 4 (GsMTx4) was tested against the multifunctional membrane protein mutant TMEM120A M207A. The FLAG-tagged TMEM120A variant was immobilized on anti-FLAG biosensors-representing a tag-based but fluorescent- and enzyme-label-free setup-and exposed to serial dilutions of GsMTx4 to measure association and dissociation rates. The protocol integrates detergent optimization using Lauryl Maltose Neopentyl Glycol (LMNG) and complementary characterization by mass photometry and differential scanning fluorimetry (DSF). This workflow enables reliable kinetic measurements and is compatible with medium-throughput screening. The approach may be applicable to other membrane protein-ligand systems where detergent stability is maintained. Overall, the study highlights key parameters influencing immobilization efficiency and analyte delivery, supporting the potential utility of BLI in inhibitor profiling and mechanistic studies of membrane protein interactions.