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

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

Simultaneous power and beam-shape optimization of an OPSL resonator

In the assembly of optical resonators of optically pumped semiconductor lasers (OPSL), the highly reflective resonator mirror is the most crucial component. In previous cooperation, Coherent and Fraunhofer IPT have developed a robust active alignment strategy to optimize the output power of the OPSL resonator using search strategies for finding the laser threshold as well as hill-climbing algorithms for maximizing the output power. Beam-shape as well as the laser mode have major influence on the quality and the duration of subsequent beam-shaping and fiber-coupling steps. Therefore, the alignment algorithm optimizing the output power has been extended recently by simultaneous image processing for ensuring a Gaussian beam as the result of alignment. The paper describes the enhanced approach of automated alignment by additionally scanning along the optical resonator and subsequently evaluating and optimizing the roundness of the beam as well as minimizing the beam radius through twisting and tilting of the mirror. A quality metric combining these measures is defined substituting an M-2 measurement. The paper also describes the approach for automated assembly including the measuring setup, micromanipulation and dispensing devices

Characterisation of subunit II and its oligomer from spinach chloroplast ATP synthase

Proton ATP synthases carry out energy conversion in mitochondria, chloroplasts, and bacteria. A key element of the membrane integral motor CFO in chloroplasts is the oligomer of subunit III: it converts the energy of a transmembrane electrochemical proton gradient into rotational movement. To enlighten prominent features of the structure-function relationship of subunit III from spinach chloroplasts, new isolation methods were established to obtain highly pure monomeric and oligomeric subunit III in milligram quantities. By Fourier-transform infrared (FTIR) and CD spectroscopy, the predominantly a-helical secondary structure of subunit III was demonstrated. For monomeric subunit III, a conformational change was observed when diluting the SDS-solubilized protein. Under the same conditions the conformation of the oligomer III did not change. A mass of 8003 Da for the monomeric subunit III was determined by MALDI mass spectrometry (MALDI-MS), showing that no posttranslational modifications occurred. By ionisation during MALDI-MS, the noncovalent homooligomer III14 disaggregated into its III monomers. (C) 2003 Elsevier B.V All rights reserved

Biophysics and Bioinformatics Reveal Structural Differences of the Two Peripheral Stalk Subunits in Chloroplast ATP Synthase

ATP synthases convert an electrochemical proton gradient into rotational movement to produce the ubiquitous energy currency adenosine triphosphate. Tension generated by the rotational torque is compensated by the stator. For this task, a peripheral stalk flexibly fixes the hydrophilic catalytic part F1 to the membrane integral proton conducting part F(O) of the ATP synthase. While in eubacteria a homodimer of b subunits forms the peripheral stalk, plant chloroplasts and cyanobacteria possess a heterodimer of subunits I and II. To better understand the functional and structural consequences of this unique feature of photosynthetic ATP synthases, a procedure was developed to purify subunit I from spinach chloroplasts. The secondary structure of subunit I, which is not homologous to bacterial b subunits, was compared to heterologously expressed subunit II using CD and FTIR spectroscopy. The content of alpha-helix was determined by CD spectroscopy to 67% for subunit I and 41% for subunit II. In addition, bioinformatics was applied to predict the secondary structure of the two subunits and the location of the putative coiled-coil dimerization regions. Three helical domains were predicted for subunit I and only two uninterrupted domains for the shorter subunit II. The predicted length of coiled-coil regions varied between different species and between subunits I and II

ATP synthase: constrained stoichiometry of the transmembrane rotor

Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

Concentration Gradient Effects of Sodium and Lithium Ions and Deuterium Isotope Effects on the Activities of H+-ATP Synthase from Chloroplasts

We explored the concentration gradient effects of the sodium and lithium ions and the deuterium isotope's effects on the activities of H+-ATP synthase from chloroplasts (CF0F1). We found that the sodium concentration gradient can drive the ATP synthesis reaction of CF0F1. In contrast, the lithium ion can be an efficient enzyme-inhibitor by blocking the entrance channel of the ion translocation pathway in CF0. In the presence of sodium or lithium ions and with the application of a membrane potential, unexpected enzyme behaviors of CF0F1 were evident. To account for these observations, we propose that both of the sodium and lithium ions could undergo localized hydrolysis reactions in the chemical environment of the ion channel of CF0. The protons generated locally could proceed to complete the ion translocation process in the ATP synthesis reaction of CF0F1. Experimental and theoretical deuterium isotope effects of the localized hydrolysis on the activities of CF0F1, and the energetics of these related reactions, support this proposed mechanism. Our experimental observations could be understood in the framework of the well-established ion translocation models for the H+-ATP synthase from Escherichia coli, and the Na+-ATP synthase from Propionigenium modestum and Ilyobacter tartaricus