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.
Subject(s)
Electron Transport Complex IV/chemistry , Lipid Bilayers/chemistry , Membrane Proteins/chemistry , Nanoparticles/chemistry , Phospholipids/chemistry , Silicon Dioxide/chemistry , Electron Transport Complex IV/metabolism , Lipid Bilayers/chemical synthesis , Models, Molecular , Paracoccus denitrificans/enzymology , Particle Size , Phospholipids/chemical synthesis , Surface PropertiesABSTRACT
As a surrogate of live cells, proteo-lipobeads are presented, encapsulating functional membrane proteins in a strict orientation into a lipid bilayer. Assays can be performed just as on live cells, for example using fluorescence measurements. As a proof of concept, we have demonstrated proton transport through cytochrome c oxidase.
Subject(s)
Electron Transport Complex IV/chemistry , Lipid Bilayers/chemistry , Sepharose/chemistry , Biomimetics , Fluorescence , Fluorescent Dyes/chemistryABSTRACT
Here we present a surface based on double-layered nanoparticle stacks suitable for spectro-electrochemical applications. The structure is formed on a continuous gold layer by a two-dimensional periodic array of stacks of gold and tantalum pentoxide nanodisks. Reflection spectra in the visible wavelength region showed the multiple-resonant nature of surface plasmon (SP) excitations in the nanostructure, which is in good agreement with simulations based on a finite-difference-time-domain method. The multiple SP resonances can be tuned to various wavelength regions, which are required for simultaneous enhancement at excitation and emission wavelengths. Cyclic voltammetry measurements on the nanostructure proved the applicability of electrochemical methods involving interfacial redox processes.
Subject(s)
Gold/chemistry , Metal Nanoparticles/chemistry , Spectrum Analysis , Electrochemistry , Ferricyanides/chemistry , Oxides/chemistry , Tantalum/chemistryABSTRACT
Proteo-lipobeads (PLBs) are investigated as cell-free model systems to encapsulate membrane proteins such as ion channels and transporters. PLBs are based on nickel nitrile tri-acetic acid (Ni-NTA)-functionalized agarose beads, onto which membrane proteins (MP) are bound via histidine(his)-tag. Composite beads thus obtained (subsequently called proteobeads) are dialyzed in the presence of lipid micelles to form PLBs. As an example we employed cytochrome c oxidase from P. denitrificans with a his-tag fused to the C-terminus of subunitI. In this orientation the P side of CcO faces the outside of the PLB and hence protons are released to the outer aqueous phase, when electron transfer is initiated by light excitation of Ru complexes. Proton release kinetics was probed by fluorescence microscopy using the pH-sensitive sensor molecule fluorescein DHPE inserted into the lipid layer. In order to monitor the generation of membrane potentials we performed a FLIPR assay on the CcO embedded in PLBs using the FRET pair CC2-DMPE/DiSBAC2(3). The combined results show that PLBs can be used as a model system designed to quantify the kinetic parameters of membrane proteins. In addition, the FLIPR assay demonstrates the feasibility of PLBs for high throughput screening applications.