title: The structural orientation of antibody layers bound to engineered biosensor surfaces.
abstract: This paper describes a membrane protein array that binds immunoglobulin G at its constant regions whilst leaving the variable regions free to bind antigen. The scaffold of the array is the transmembrane domain of outer membrane protein A (tOmpA) from Escherichia coli engineered to assemble as an oriented monolayer on gold surfaces via a single cysteine residue. Other protein domains can be fused to the N and C termini of the scaffold. In this study we use circularly permuted ctOmpA fused to two Z domains of Staphylococcus aureus protein A (ZZctOmpA) to create the immunoglobulin G-binding array. The solution structure of the engineered proteins was assessed by circular dichroism spectroscopy. Assembly of the array, attachment of antibodies and antigen binding were measured using surface plasmon resonance and neutron reflection. Compared to mouse IgG2, polyclonal IgG from rabbit bound very strongly to ZZctOmpA and the dissociation of the immunoglobulin was slow enough to allow neutron reflection studies of the assembled layer with antigen. Using both magnetic and isotopic contrasts a complete layer by layer model was defined which revealed that the 223 Å high layer contains antibodies in an upright orientation.
authors.affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Anton P
authors.initials: AP
authors.lastname: Le Brun
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Holt
authors.forename: Deepan S H
authors.initials: DS
authors.lastname: Shah
authors.forename: Charles F
authors.initials: CF
authors.lastname: Majkrzak
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002942:Circular Dichroism; D017666:Deuterium Oxide / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013499:Surface Properties
chemical_list: D000906:Antibodies; D000941:Antigens; D001425:Bacterial Outer Membrane Proteins; D055767:Immobilized Proteins; D007074:Immunoglobulin G; C078167:OMPA outer membrane proteins; D006046:Gold; D017666:Deuterium Oxide