Stable self-assembly of a protein engineering scaffold on gold surfaces.
The outer membrane protein OmpF from Escherichia coli is a member of a large family of beta-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 beta-mercaptoethanol and thiolipids are added after the protein immobilisation step, the protein is shown, by Fourier transform infrared spectroscopy (FTIR), to retain its beta-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 beta-barrel proteins are recognized protein engineering scaffolds, the method provides a generic method for the simple self-assembly of protein interfaces from aqueous solution.
Terrettaz
Samuel
S
Institute of Biomolecular Sciences, Swiss Federal Institute of Technology Lausanne, Switzerland.
Ulrich
Wolf-Peter
WP
Vogel
Horst
H
Hong
Qi
Q
Dover
Lynn G
LG
Lakey
Jeremy H
JH
D000818:Animals; D001665:Binding Sites; D003087:Colicins / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000138:chemical synthesis; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared; D020349:Surface Plasmon Resonance; D013499:Surface Properties
D003087:Colicins; D008051:Lipid Bilayers; C109237:OmpF protein; D018272:Porins; D011994:Recombinant Proteins; D006046:Gold; D003545:Cysteine