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

Abstract

The outer membrane protein OmpF from Escherichia coli is a member of a large family of ␤-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 ␤-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its ␤-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 ␤-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.

Two-step Membrane Binding by Equinatoxin II, a Pore-forming Toxin from the Sea Anemone, Involves an Exposed Aromatic Cluster and a Flexible Helix

“…1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Fluka. Thiolipids, DP-mercapto-PA (O-(8Ј-mercapto-3Ј,6Ј-dioxaoctyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid), were a gift from Horst Vogel (Lausanne, Switzerland) (29,30). Octyl-POE was purchased from Alexis Biochemicals (San Diego, CA).…”

A generic expression system to produce proteins that co-assemble with alkane thiol SAM

“…We have previously shown that by pre-treating the surface with β-mercapto-ethanol surface interactions are reduced and proteins keep their 3D structure (Terrettaz et al 2002;Keegan et al 2005) the subsequent assembly with lipids stabilizes the array and further contributes to retaining protein structure (Terrettaz et al 2002;Holt et al 2005;Cisneros et al 2006). This expression system has the potential to increase the range and complexity of proteins that can be specifi cally immobilized on surfaces designed to modify cellular growth (Shah et al 2007) or the development of new detector elements for SPR, SAW, or QCM.…”

“…The specifi c immobilization of proteins upon surfaces has the potential to revolutionize both the study of their natural properties and their utilization in novel, selfassembling nanostructures (Terrettaz et al 2002). Patterned proteins have applications in molecular biosensors and protein arrays (Bertone and Snyder 2005), but can also contribute to cell patterning for research (Liu et al 2005;Sniadecki et al 2006) tissue engineering and cell-based biosensors (Csucs et al 2003;Hasirci and Kenar 2006;Petty et al 2007;Yap and Zhang 2007).…”

“…A simple and widely applicable immobilization strategy of large proteins with SAM has yet to be developed. Two of the best methods come from the Mrksich group who has developed a fusion protein system with chitninase fusion proteins binding to SAM immobilized chitin analogues (Hodneland et al 2002) or a cysteine-directed immobilization into mixed maleimide and ethylene glycol terminated SAM (Houseman et al 2003) An alternative approach is to fuse the protein of interest to a membrane protein scaffold which self assembles with SAM (Terrettaz et al 2002;Shah et al 2007). It has also been shown that combined surfaces of lipid and protein could provide patterned arrays in which the nonprotein surface had extremely low nonspecifi c binding due to the properties of lipid layers (Kung et al 2000).…”

Semicontinuous Measurement of Mite Allergen (Der f 2) Using a Surface Acoustic Wave Immunosensor under Moderate pH for Long Sensor Lifetime

“…(18)(19)(20) The surface modification of the sensing area with the SAM was carried out as per the manufacturer's protocol: the surface was initially rendered hydrophilic in order to prevent nonspecific hydrophobic adsorption of ORLA85 by incubating the sensing area with 1% (v/v) BME in ultrapure water for 5 min. (21) Afterwards, in order to form the Au-S bond between ORLA85 and the gold on the SAW device, disulfide bonds of ORLA85 were reduced by mixing the protein solution with TCEP solution (TCEP 7.5 mg/mL in Tris-HCl) and letting stand for 30 min. Then the mixed solution was applied to the sensing area, and incubated for 10 min to form the SAM.…”