title: Silica nanoparticles for the oriented encapsulation of membrane proteins into artificial bilayer lipid membranes.
abstract: An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.
authors: Austrian Institute of Technology GmbH , AIT, Donau-City Str. 1, 1220 Vienna, Austria.
authors: Florian
authors: F
authors: Schadauer
authors: Andreas F
authors: AF
authors: Geiss
authors: Johannes
authors: J
authors: Srajer
authors: Bernhard
authors: B
authors: Siebenhofer
authors: Pinar
authors: P
authors: Frank
authors: Ciril
authors: C
authors: Reiner-Rozman
authors: Bernd
authors: B
authors: Ludwig
authors: Oliver-M H
authors: OM
authors: Richter
authors: Christoph
authors: C
authors: Nowak
authors: Renate L C
authors: RL
authors: Naumann
mesh_terms: D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers / Q000138:chemical synthesis / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D053758:Nanoparticles / Q000737:chemistry*; D010231:Paracoccus denitrificans / Q000201:enzymology; D010316:Particle Size; D010743:Phospholipids / Q000138:chemical synthesis / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D012822:Silicon Dioxide; D003576:Electron Transport Complex IV