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Stable self-assembly of a protein engineering scaffold on gold surfaces

¹ Institute of Biomolecular Sciences, Swiss Federal Institute of Technology Lausanne, CH-1015 Lausanne, Switzerland
² School of Biochemistry and Genetics, The Medical School, Newcastle upon Tyne, NE2 4HH UK

Reprint requests to: Jeremy H. Lakey, School of Biochemistry and Genetics, The Medical School, Newcastle upon Tyne, NE2 4HH UK; e-mail: j.h.lakey{at}ncl.ac.uk; fax: (44)01912227424.

The outer membrane protein OmpF from Escherichia coli is a member of a large family of ß-barrel membrane proteins. Some, like OmpF, are pore-forming proteins whilse others are active transporters or enzymes. We have previously shown that the receptor-binding domain (R-domain) of the toxin colicin N binds with high affinity to OmpF reconstituted into tethered lipid bilayers on gold electrodes. The binding can be measured by surface plasmon resonance (SPR) and ion channel blockage (impedance spectroscopy, IS). In this paper we report the use of a mutant OmpF-E183C in which a single cysteine had been introduced on a short periplasmic turn. OmpF-E183C binds directly to gold surfaces and creates high-density protein layers by self-assembly from detergent solution. When the gold surface is pretreated with ß-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its ß-rich structure. Furthermore, we could also measure R-domain binding by SPR and IS, confirming the functional reconstitution of a self-assembled membrane protein monolayer at the gold surface. Because these ß-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.

Keywords: Outer membrane protein; tethered lipid bilayer; FTIR; surface plasmon resonance; impedance spectroscopy; nanotechnology

Abbreviations: DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine • DphyPC: 1,2-diphytanoyl-sn-glycero-3-phosphocholine • DP-mercapto-PA: O-(8'-mercapto-3',6'-dioxaoctyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid • DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine • DTT: dithiothreitol • FTIR: Fourier transform infrared spectroscopy • IS: impedance spectroscopy • Omp: outer membrane protein • OPOE: n-Octyl-polyoxyethylene • RA: reflectance absorbance • R-domain: receptor binding domain • SDS: sodium dodecyl sulfate • SPR: surface plasmon resonance

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