Silica nanoparticles for the oriented encapsulation of membrane proteins into artificial bilayer lipid membranes.

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.

Proteo-lipobeads for the oriented encapsulation of membrane proteins.

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.

Transient absorption spectroscopy on spiropyran monolayers using nanosecond pump-probe Brewster angle reflectometry.

Self-assembled monolayers of 11-(3',3'-dimethyl-6,8-dinitrospiro[chromene-2,2'-indoline]-1'-yl) undecanoic acid (amphiphilic spiropyran) at the air-water interface are studied using Brewster angle reflectometry. Transient kinetics of the spiropyran to merocyanine conversion are recorded in a UV-pump, VIS-probe configuration. By varying the probe wavelength using an optical parametric oscillator, we are able to reconstruct absorption spectra of intermediate states with a time-resolution of 10 nanoseconds, limited by the temporal convolution of the two laser pulses. After UV irradiation, spiropyran converts to merocyanine in two stages. The first occurs within a timescale of several tens of nanoseconds and is heavily convoluted with the system response time, whereas the second stage occurs over a few hundred nanoseconds. During the rise time there is a small red shift in the transient absorption spectrum of ~20 nm. We assign the red shift and the slower kinetics to the isomerization of a merocyanine isomer cis about the central methine bond to those that are trans about the same bond.

Fingerprints of single nuclear spin energy levels using STM - ENDOR.

We performed STM-ENDOR experiments where the intensity of one of the hyperfine components detected in ESR-STM is recorded while an rf power is irradiated into the tunneling junction and its frequency is swept. When the latter frequency is near a nuclear transition a dip in ESR-STM signal is observed. This experiment was performed in three different systems: near surface SiC vacancies where the electron spin is coupled to a next nearest neighbor 29Si nucleus; Cu deposited on Si(111)7x7 surface, where the unpaired electron of the Cu atom is coupled to the Cu nucleus (63Cu, 65Cu) and on Tempo molecules adsorbed on Au(111), where the unpaired electron is coupled to a Nitrogen nucleus (14N). While some of the hyperfine values are unresolved in the ESR-STM data due to linewidth we find that they are accurately determined in the STM-ENDOR data including those from remote nuclei, which are not detected in the ESR-STM spectrum. Furthermore, STM-ENDOR can measure single nuclear Zeeman frequencies, distinguish between isotopes through their different nuclear magnetic moments and detect quadrupole spectra. We also develop and solve a Bloch type equation for the coupled electron-nuclear system that facilitates interpretation of the data. The improved spectral resolution of STM - ENDOR opens many possibilities for nanometric scale chemical analysis.