Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection.
Protein arrays are used in a wide range of applications. The array described here binds IgG antibodies, produced in rabbit, to gold surfaces via a scaffold protein. The scaffold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron Reflection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deuterium exchange in the biological layer. Polarised Neutron Reflection allows for the modelling of many and complex layers with good fits. The total thickness of the biological layer immobilised on the gold surface is found to be 187 A and the layer can thus far be separated into its lipid, protein and solvent parts.
Le Brun
Anton P
AP
Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. anton.le-brun@ncl.ac.uk
Holt
Stephen A
SA
Shah
Deepan S
DS
Majkrzak
Charles F
CF
Lakey
Jeremy H
JH
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D006046:Gold / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D008280:Magnetics; D009502:Neutrons; D040081:Protein Array Analysis; D013499:Surface Properties
D001425:Bacterial Outer Membrane Proteins; D007074:Immunoglobulin G; C078167:OMPA outer membrane proteins; D006046:Gold