title: Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy.
abstract: Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 10(6) M(-1)·s(-1) at 0.43 μM PnA to (1.1 ± 0.1) × 10(5) M(-1)·s(-1) at 12 μM PnA. CTB-GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 10(9) M(-1)·s(-1) at 0.5 nM CTB to (3.5 ± 0.2) × 10(6) M(-1)·s(-1) at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 10(9) M(-1) and (2.8 ± 0.5) × 10(13) M(-1), respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB-GM1 demonstrates positive ligand-ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein-ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Room 2020, Salt Lake City, Utah 84112, United States.
authors.forename: Krystal L
authors.initials: KL
authors.lastname: Sly
authors.forename: John C
authors.initials: JC
authors.lastname: Conboy
mesh_terms: D001665:Binding Sites; D002772:Cholera Toxin / Q000032:analysis* / Q000737:chemistry*; D005997:Glycerylphosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry*; D019887:Peanut Agglutinin / Q000032:analysis* / Q000737:chemistry*; D010713:Phosphatidylcholines; D011485:Protein Binding; D013057:Spectrum Analysis
chemical_list: D008024:Ligands; D008051:Lipid Bilayers; D019887:Peanut Agglutinin; D010713:Phosphatidylcholines; D005997:Glycerylphosphorylcholine; D002772:Cholera Toxin; C017251:1,2-oleoylphosphatidylcholine