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

Area of Science:

• Biophysics
• Biochemistry
• Materials Science

Background:

• Measuring high-affinity protein-membrane interactions is crucial but challenging.
• Existing methods often require labels or large sample volumes, limiting sensitivity.
• Liposome-protein interactions are fundamental to cellular processes and disease.

Purpose of the Study:

• To develop an ultrasensitive, direct detection method for protein binding to liposome membranes.
• To quantify the binding affinity of Perfringolysin O (PFO) to cholesterol-rich liposomes.
• To demonstrate a label-free, noninvasive approach for probing protein-membrane interactions.

Main Methods:

• Utilized high-throughput second harmonic scattering (SHS) for signal detection.
• Employed Perfringolysin O (PFO) as a model protein selectively binding to cholesterol-rich membranes.
• Used large unilamellar vesicles (LUVs) with varying cholesterol concentrations.

Main Results:

• Achieved ultrasensitive, direct detection of PFO binding to liposomes.
• Quantified PFO binding to cholesterol-rich membranes (cholesterol >30%) with a dissociation constant (Kd) of (1.5 ± 0.2) × 10^-12 M.
• Demonstrated no significant SHS signal or pore formation at 20% cholesterol concentration.

Conclusions:

• High-throughput SHS is a powerful, label-free technique for studying protein-membrane interactions.
• The method enables detection of interactions at sub-picomolar concentrations with ultrasmall sample volumes (<10 μL).
• This approach facilitates the study of low-abundance proteins and their membrane interactions, advancing biophysical and biochemical research.