IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance.
Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.
Mitomo
Hideyuki
H
Frontier Collaboration Research Center, Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuda, Midori-ku, Yokohama 226-8501, Japan.
Shigematsu
Hideki
H
Kobatake
Eiry
E
Furusawa
Hiroyuki
H
Okahata
Yoshio
Y
D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011791:Quartz / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism*
D007074:Immunoglobulin G; D008051:Lipid Bilayers; D013205:Staphylococcal Protein A; D011791:Quartz