79 research outputs found
From protons to OXPHOS supercomplexes and Alzheimer's disease: Structure–dynamics–function relationships of energy-transducing membranes
AbstractBy the elucidation of high-resolution structures the view of the bioenergetic processes has become more precise. But in the face of these fundamental advances, many problems are still unresolved. We have examined a variety of aspects of energy-transducing membranes from large protein complexes down to the level of protons and functional relevant picosecond protein dynamics. Based on the central role of the ATP synthase for supplying the biological fuel ATP, one main emphasis was put on this protein complex from both chloroplast and mitochondria. In particular the stoichiometry of protons required for the synthesis of one ATP molecule and the supramolecular organisation of ATP synthases were examined. Since formation of supercomplexes also concerns other complexes of the respiratory chain, our work was directed to unravel this kind of organisation, e.g. of the OXPHOS supercomplex I1III2IV1, in terms of structure and function. Not only the large protein complexes or supercomplexes work as key players for biological energy conversion, but also small components as quinones which facilitate the transfer of electrons and protons. Therefore, their location in the membrane profile was determined by neutron diffraction. Physico-chemical features of the path of protons from the generators of the electrochemical gradient to the ATP synthase, as well as of their interaction with the membrane surface, could be elucidated by time-resolved absorption spectroscopy in combination with optical pH indicators. Diseases such as Alzheimer's dementia (AD) are triggered by perturbation of membranes and bioenergetics as demonstrated by our neutron scattering studies
HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae
Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase
Mitochondria-Specific Accumulation of Amyloid β Induces Mitochondrial Dysfunction Leading to Apoptotic Cell Death
Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ1–42 treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ1–42-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ1–42 in HT22 cells using Aβ1–42 with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ1–42-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ1–42 accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ1–42-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ1–42 accumulation, which mimics the apoptosis process in exogenous Aβ1–42-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ1–42 accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations
Upconversion-induced heat generation and thermal lensing in Nd:YLF and Nd:YAG
We investigate the influence of interionic upconversion between neighboring ions in the upper laser level of Nd:YLF and Nd:YAG on population dynamics, heat generation, and thermal lensing under lasing and non-lasing conditions. It is shown that cascaded multiphonon relaxations following each upconversion process generate significant extra heat dissipation in the crystal under non-lasing compared to lasing conditions. Owing to the unfavorable temperature dependence of thermal and thermo-optical parameters, this leads, firstly, to a significant temperature increase in the rod, secondly, to strong thermal lensing with pronounced spherical aberrations and, ultimately, to rod fracture in a high-power end-pumped system. In a three-dimensional finite-element calculation, excitation densities, upconversion rates, heat generation temperature profiles, and thermal lensing are calculated. Differences in thermal lens power between non-lasing and lasing conditions up to a factor of six in Nd:YLF and up to a factor of two in Nd:YAG are experimentally observed and explained by the calculation. This results in a strong deterioration in performance when operating these systems in a Q-switched regime, as an amplifier, or on a low-gain transition. Methods to decrease the influence of interionic upconversion are discussed. It is shown that tuning of the pump wavelength can significantly alter the rod temperature
Dye removal, catalytic activity and 2D crystallization of chloroplast H+-ATP synthase purified by blue native electrophoresis
AbstractThe proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CFOF1) and its subcomplexes CFO and CF1 were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569–575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CFOF1, CFO or CF1 and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF1 and ATP synthesis activity of reconstituted CFOF1 were determined before and after dye removal. The secondary structure of CFO was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CFOF1 and CF1 nor affects the subunit composition and the high α-helical content of CFO. In crystallization attempts, 2D arrays of CFOF1 and of CFO before and after dye removal were obtained. In the aggregates of CFO, circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization
Dye-ligand chromatographic purification of intact multisubunit membrane protein complexes: application to the chloroplast H+-FoF1-ATP synthase.
n-Dodecyl-beta-D-maltoside was used as a detergent to solubilize the ammonium sulphate precipitate of chloroplast F(O)F(1)-ATP synthase, which was purified further by dye-ligand chromatography. Upon reconstitution of the purified protein complex into phosphatidylcholine/phosphatidic acid liposomes, ATP synthesis, driven by an artificial DeltapH/Deltapsi, was observed. The highest activity was achieved with ATP synthase solubilized in n-dodecyl-beta-D-maltoside followed by chromatography with Red 120 dye. The optimal dye for purification with CHAPS was Green 5. All known subunits were present in the monodisperse proton-translocating ATP synthase preparation obtained from chloroplasts
Detergent effect on anion exchange perfusion chromatography and gel filtration of intact chloroplast H+-ATP synthase
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