College Park, Maryland June 6 - 10 , 2004 |
M3-B2 (2:00 PM): Self Assembly of Membrane Protein Arrays
Stephen A. Holt (ISIS, Rutherford Appleton Laboratory), Jeremy H. Lakey, Neil Keegan (School of Cell and Molecular Biosciences, The Medical School, The University of Newcastle-upon-Tyne, NE2 4HH, United Kingdom), Sofian M. Daud (Institute for Nanoscale Science and Technology, The University of Newcastle-upon-Tyne, NE2 4HH, United Kingdom)
High density protein arrays on solid surfaces are becoming an increasingly important component of post-genomic technologies where there is a need for protein recognition interfaces in physical devices. Uses for these layers range from protein array technology for screening of the proteome through diagnostic "point of care" devices aimed at specific protein or metabolite targets to biocompatible surfaces for nanoscale design of implant surfaces or cell culture environments. DNA arrays have become well established in the marketplace, however DNA recognition is only a one-dimensional interaction. Protein-protein recognition usually relies upon the three-dimensional fold of both components. The maintenance of tertiary structure with the correct orientation relative to the substrate is essential for a well defined protein array. The outer membrane pore forming protein OmpF from Escherichia coli has been used to produce protein arrays on silicon blocks that have been coated with titanium and gold. The protein has been mutated by the addition of a single cysteine residue which enables it to chemisorb to the gold substrate with the required orientation. A thiolipid is then added to the sample cell to complete the self assembling membrane mimetic at the interface. We have applied neutron reflectometry to this system to validate our models which incorporate the relative distribution of the lipids and proteins, the exclusion of water from regions of the bilayer and the overall thickness of the lipid layers and the protein. Initial modelling of the data demonstrates that the protein array has self assembled with the expected physical dimensions. Further detailed analysis of the data is currently underway.
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