TY - JOUR
T1 - Silica Nanoparticles for the Oriented Encapsulation of Membrane Proteins into Artificial Bilayer Lipid Membranes
AU - Schadauer, Florian
AU - Geiss, Andreas
AU - Srajer, Johannes
AU - Siebenhofer, Bernhard
AU - Frank, Pinar
AU - Rozman, Ciril
AU - Ludwig, Bernd
AU - Richter, Oliver-M.H.
AU - Nowak, Christoph
AU - Naumann, Renate L. C.
PY - 2015/2/10
Y1 - 2015/2/10
N2 - 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. denitrif icans 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.
AB - 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. denitrif icans 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.
U2 - 10.1021/la504417j
DO - 10.1021/la504417j
M3 - Article
SN - 0743-7463
JO - Langmuir
JF - Langmuir
ER -