We reconstruct spectra of secondary X-rays from a tunable 250-350 MeV laser wakefield electron accelerator from single-shot X-ray depth-energy measurements in a compact (7.5 × 7.5 × 15 cm), modular ...X-ray calorimeter made of alternating layers of absorbing materials and imaging plates. X-rays range from few-keV betatron to few-MeV inverse Compton to > 100 MeV bremsstrahlung emission, and are characterized both individually and in mixtures. Geant4 simulations of energy deposition of single-energy X-rays in the stack generate an energy-vs-depth response matrix for a given stack configuration. An iterative reconstruction algorithm based on analytic models of betatron, inverse Compton and bremsstrahlung photon energy distributions then unfolds X-ray spectra, typically within a minute. We discuss uncertainties, limitations and extensions of both measurement and reconstruction methods.
Photosynthetic reaction centers (RCs) from Rb. sphaeroides with a genetically engineered 7-his-tag at the C-terminus of the M-subunit are bound to a Ni-NTA-modified gold surface. Subsequently, the ...bound RCs are subjected to in situ dialysis in the presence of lipid micelles to form a protein-tethered lipid bilayer membrane (ptBLM). Redox properties of the RC thus immobilized are investigated by cyclic voltammetry. Photocurrrents are generated in the range of 10 μA cm–2, however, different from previous studies at potentials of −200 and −300 mV, and without cytochrome c as a mediator. The unexpected behavior is explained in terms of an interprotein reaction between RC molecules promoted by the lipid bilayer, which we had previously detected by surface-enhanced infrared absorption spectroscopy.
A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering ...molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome
c oxidase (C
cO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.
Time-resolved surface-enhanced IR-absorption spectroscopy triggered by electrochemical modulation has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. Single bands isolated from a ...broad band in the amide I region using phase-sensitive detection were attributed to different redox centers. Their absorbances changing on the millisecond timescale could be fitted to a model based on protonation-dependent chemical reaction kinetics established previously. Substantial conformational changes of secondary structures coupled to redox transitions were revealed.
Biomimetic Membranes for Multi-Redox Center Proteins Naumann, Renate L C; Geiss, Andreas F; Steininger, Christoph ...
International journal of molecular sciences,
03/2016, Volume:
17, Issue:
3
Journal Article
Peer reviewed
Open access
His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs). An overview is presented on various surfaces ranging from flat to spherical and modified with ...linker molecules with nitrile-tri-acetic acid (NTA) terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM). MRPs, such as the cytochrome c oxidase (CcO) from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs) from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced resonance Raman spectroscopy (SERRS), were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs). The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM) and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.
Multigap Resistive Plate Chambers (MRPCs) are often used as time-of-flight (TOF) detectors for high-energy physics and nuclear experiments thanks to their excellent time accuracy. For the Compressed ...Baryonic Matter (CBM) TOF system, MRPCs are required to work at particle fluxes on the order of 1–10kHz/cm2 for the outer region and 10–25kHz/cm2 for the central region. Better time resolution will allow particle identification with TOF techniques to be performed at higher momenta. From our previous studies, a time resolution of 25ps has been obtained with a 20-gap MRPC of 140μm gap size with enhanced rate capability. By using a new type of commercially available thin low-resistivity glass, further improvement MRPC rate capability is possible. In order to study the rate capability of the 10-gap MRPC built with this new low-resistivity glass, we have performed tests using the continuous electron beam at ELBE. This 10-gap MRPC, with 160μm gaps, reaches 97% efficiency at 19.2kV and a time resolution of 36ps at particle fluxes near 2kHz/cm2. At a flux of 100kHz/cm2, the efficiency is still above 95% and a time resolution of 50ps is obtained, which would fulfil the requirement of CBM TOF system.
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.
His-tag technology is employed to bind membrane proteins, such as the bc 1 complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict ...orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 μm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc 1 activated by the RC. To assess the turnover of bc 1 excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.
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