Ultrasensitive Label-Free Detection of Protein–Membrane Interaction Exemplified by Toxin-Liposome Insertion

• Published in: The journal of physical chemistry letters 2022-04, Vol.13 (14), p.3197-3201
• Main Authors: Schönfeldová, T, Okur, H. I, Vezočnik, V, Iacovache, I, Cao, C, Dal Peraro, M, Maček, P, Zuber, B, Roke, S
• Format: Article
• Language: English
• Subjects: Bacterial Toxins - metabolism; Cholesterol - metabolism; Cryoelectron Microscopy; Hemolysin Proteins - metabolism; Letter; Physical Insights into Chemistry, Catalysis, and Interfaces; Protein Binding - physiology; Second Harmonic Generation Microscopy; Unilamellar Liposomes - metabolism
• ISSN: 1948-7185; 1948-7185
• DOI: 10.1021/acs.jpclett.1c04011

Measuring the high-affinity binding of proteins to liposome membranes remains a challenge. Here, we show an ultrasensitive and direct detection of protein binding to liposome membranes using high throughput second harmonic scattering (SHS). Perfringolysin O (PFO), a pore-forming toxin, with a highly membrane selective insertion into cholesterol-rich membranes is used. PFO inserts only into liposomes with a cholesterol concentration >30%. Twenty mole-percent cholesterol results in neither SHS-signal deviation nor pore formation as seen by cryo-electron microscopy of PFO and liposomes. PFO inserts into cholesterol-rich membranes of large unilamellar vesicles in an aqueous solution with K d = (1.5 ± 0.2) × 10–12 M. Our results demonstrate a promising approach to probe protein–membrane interactions below sub-picomolar concentrations in a label-free and noninvasive manner on 3D systems. More importantly, the volume of protein sample is ultrasmall (