Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy

Area of Science:

• Biophysics
• Molecular Interactions
• Spectroscopy

Background:

• Multivalent proteins like peanut agglutinin (PnA) and cholera toxin B subunit (CTB) play crucial roles in biological processes and diagnostics.
• Understanding the binding kinetics of these proteins to lipid bilayers is essential for developing advanced biosensors and immunoassays.
• Investigating protein-ligand interactions at the molecular level provides insights into complex biological systems.

Purpose of the Study:

• To investigate the binding kinetics of PnA and CTB proteins to a GM1-doped lipid bilayer using second-harmonic correlation spectroscopy (SHCS).
• To determine adsorption and desorption rates, binding affinity, and binding free energy for these protein-lipid interactions.
• To compare SHCS findings with traditional equilibrium binding isotherm methods and analyze protein-ligand cooperativity.

Main Methods:

• Utilized second-harmonic correlation spectroscopy (SHCS) to measure binding kinetics.
• Employed traditional equilibrium binding isotherm analysis.
• Investigated protein-ligand interactions at varying bulk protein concentrations (PnA: 0.43–12 μM; CTB: 0.5–240 nM).

Main Results:

• SHCS determined adsorption and desorption rates, revealing that adsorption rates decreased with increasing protein concentration for both PnA and CTB.
• Desorption rates showed no dependence on initial protein concentration for either protein.
• Strongest binding affinities were observed at the lowest concentrations: (3.7 ± 0.8) × 10^9 M⁻¹ for PnA and (2.8 ± 0.5) × 10^13 M⁻¹ for CTB.
• Analysis indicated electrostatic repulsion for PnA-GM1 binding and positive ligand-ligand cooperativity for CTB-GM1 interactions.

Conclusions:

• SHCS is a powerful technique for examining complex protein-ligand interactions in lipid bilayers.
• The study provides valuable kinetic data and insights into the binding mechanisms of multivalent proteins.
• Findings contribute to the understanding of molecular interactions relevant to biosensors, immunoassays, and biomedical diagnostics.