David Moss

Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein.

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90–232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90–232 showed an infrared absorbance spectrum typical of proteins with a predominant α-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3–2 m guanidine hydrochloride), the secondary structure of SHaPrP90–232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel β-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in α-helical structures and the increase in β-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No β-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90–232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as “critical oligomers.” The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90–232. A new model for the structural transition and aggregation process of the prion protein is described.

CYCLIC ELECTRON TRANSPORT IN CHLOROPLASTS THE Q-CYCLE AND THE SITE OF ACTION OF ANTIMYCIN

Cyclic electron transport systems have been set up in broken chloroplasts, with photochemically reduced ferredoxin or 9,10-anthraquinone-2-sulphonate as cofactor. In good agreement with the literature, only the ferredoxin-catalyzed pathway was found to be inhibited by antimycin; but both pathways were found to have a slow electrogenic reaction, both were inhibited by the cytochrome b-563 oxidation inhibitor 2-heptyl-4-hydroxyquinoline N-oxide (the inhibition being strongest at limiting light intensity), and the two pathways had the same proton/electron stoichiometry at limiting light intensity. It is concluded that a Q-cycle can occur in cyclic electron transport with either cofactor; and therefore that the site of action of antimycin in chloroplasts is not within the Q-cycle, as it is believed to be in mitochondria and bacteria. Instead, a ferredoxin-quinone reductase is proposed as the site of action of antimycin in the ferredoxin-catalyzed cyclic pathway. It is also concluded that the data presented here are consistent with the suggestion that the Q-cycle in photosynthetic electron transport is a facultative one, its degree of engagement depending on competition between the Rieske centre and cytochrome b-563 for reducing equivalents from plastosemiquinone.

The interactions of duroquinol, DBMIB and NQNO with the chloroplast cytochrome bf complex

Duroquinol reduces the endogenous plastoquinone pool of chloroplasts only very slowly because of a lack of quinol-quinone transhydrogenase activity of the cytochrome bf complex, but is a fast direct donor into the cytochrome bf complex via the quinol oxidation site. It also accelerates the rate of cytochrome b -563 reoxidation in the presence of NQNO. Results of three independent methods are consistent with a requirement of one DBMIB per bf monomer for full inhibition of bf complex electron transfer activity, demonstrating that a bf complex monomeric unit is capable of electron transfer activity. At low molar ratios, inhibition reverses in the dark but is fully restored after several turnovers of the complex. Inhibition is weakened by high concentrations of duroquinol or decyl -plastoquinol. Free DBMIB is rapidly reduced to DBMIBH 2 by duroquinol. Addition of DBMIBH 2 in the dark does not cause a large inhibition of the first turnover of the bf complex. It is proposed that DBMIBH 2 does not form a stable inhibitory complex but can be oxidised by the quinol oxidation site of the cytochrome bf complex, but at a rate which is significantly less than that of oxidation of plastoquinol or duroquinol. The tight inhibitory complex involves the semiquinone or quinone form of DBMIB and this bound species is only slowly reduced in the dark back to free DBMIBH 2 . Subsequent flash oxidation leads to a competition between DBMIBH 2 , duroquinol and plastoquinol for the quinol oxidation site. NQNO and HQNO are effectors of the quinone reduction site of the chloroplast cytochrome bf complex. In the analogous mitochondrial bc 1 complex, HQNO inhibits electron transfer through the quinone reduction site and this inhibition is not simply a result of the modulation of the midpoint potential of the cytochrome b H haem. In the cytochrome bf complex, a large increase of the midpoint potentials of the cytochrome b haems has been reported, but we were unable to repeat this observation. Although the extent of oxidant-induced reduction of haems b is increased by N(H)QNO, inhibition of electron transfer through the quinone reduction site is small, especially in the presence of duroquinol, and the electrogenic reaction which is associated with protonmotive function of the enzyme is not impaired even after multiple turnovers. It is proposed that the Q i site of the cytochrome bf complex can still turn over sufficiently rapidly in the presence of NQNO or HQNO so that there is little inhibition of turnover, even although transient kinetic behaviour can be markedly affected.

Kinetic studies of electron transfer in a hybrid system constructed from the cytochrome bf complex and Photosystem I

Abstract (1) A hybrid electron-transfer system has been constructed from purified cytochrome bf complex and Photosystem I preparations which are active in plastocyanin oxidation. Donor and acceptor characteristics of these purified preparations are described. (2) The use of this simplified system enabled the application of a rigorous method of component deconvolution from spectral kinetic data. This relied on matrix analysis of the extinction coefficients of all components at each wavelength of measurement. (3) Kinetic analyses of flash-induced redox changes of cytochromes b -563, cytochrome f , plastocyanin and P-700 were made with this method and were compared with computer simulations of predicted behaviour. These studies indicated that cytochrome b -563 behaviour was consistent with its being reduced by ‘oxidant-induced reduction’ at centre o, even although cytochrome b -563 reduction was apparently much faster than cytochrome f rereduction after a flash. However, in order to obtain good agreement between experiment and simulation, the midpoint potential of the Rieske centre had to be assumed to be +370 mV. (4) Multiple turnover behaviour of the cytochrome bf complex at high potentials was also consistent with the Q-cycle feature of oxidant-induced reduction of cytochrome b . However, a maximum reduction of only around 25% of total cytochrome b -563 could be achieved via this route, and this extent was neither affected by 2- n -nonyl-4-hydroxyquinoline N -oxide (NQNO) nor by added quinone. Similarly, in thylakoids in the presence of NQNO, less than half of the total cytochrome b -563 could be reduced by multiple flashes. (5) Continuous illumination experiments at a low potential indicated that cytochrome b oxidation occurred on turnover of the cytochrome bf complex even when the cytochromes b were fully prereduced. The oxidation was sensitive to NQNO.

The supramolecular organization of self-assembling chlorosomal bacteriochlorophyll c, d, or e mimics

Bacteriochlorophylls (BChls) c, d, and e are the main light-harvesting pigments of green photosynthetic bacteria that self-assemble into nanostructures within the chlorosomes forming the most efficient antennas of photosynthetic organisms. All previous models of the chlorosomal antennae, which are quite controversially discussed because no single crystals could be grown so far from these organelles, involve a strong hydrogen-bonding interaction between the 31 hydroxyl group and the 131 carbonyl group. We have synthesized different self-assemblies of BChl c mimics having the same functional groups as the natural counterparts, that is, a hydroxyethyl substituent, a carbonyl group and a divalent metal atom ligated by a tetrapyrrole. These artificial BChl mimics have been shown by single crystal x-ray diffraction to form extended stacks that are packed by hydrophobic interactions and in the absence of hydrogen bonding. Time-resolved photoluminescence proves the ordered nature of the self-assembled stacks. FT-IR spectra show that on self-assembly the carbonyl frequency is shifted by ≈30 cm−1 to lower wavenumbers. From the FT-IR data we can infer the proximal interactions between the BChls in the chlorosomes consistent with a single crystal x-ray structure that shows a weak electrostatic interaction between carbonyl groups and the central zinc atom.

Electrochemical redox titration of cofactors in the reaction center from Rhodobacter sphaeroides

The electrochemical redox poising of the primary electron donor P and of the quinone electron acceptor(s) Q in isolated reaction centers from Rhodobacter sphaeroides in an ultra‐thin‐layer electrochemical cell, monitored by chronoamperometry and by spectroscopy in the visible/near‐infrared region, is reported. Electrical application of a redox potential of +0.4 V (vs. Ag/AgCl/3 M KCl) leads to quantitative formation of the π‐cation radical of P within a few minutes. The oxidized product can be re‐reduced to the neutral species by application of 0 V, and full reversibility is maintained over many‐cycles. By poising at a series of intermediate potentials, a titration curve for the 865 nm P band was obtained, which could be fitted to a Nernst function with E m = 0.485 vs. SHE and n = 0.96. By Application of negative potentials (−0.2 V and −0.45 V vs. Ag/AgCl/3 M KCl), the quinone electron acceptors were reversibly reduced as demonstrated by the shift of bacteriopheophytin absorption and drastically changed kinetics of charge recombination. The use of this thin‐layer electrochemical technique for the determination of midpoint potentials, for the investigation of redox‐poised electron transfer reactions as well as for spectroscopy in the mid‐infrared region is discussed.

Circulating fibronectin affects bone matrix, whereas osteoblast fibronectin modulates osteoblast function

The bone matrix is composed mostly of collagen, but the initial and continuous presence of fibronectin was found to be crucial for collagen matrix integrity in vitro. It has been assumed that osteoblasts produce the fibronectin required for bone matrix formation. Using transgenic mice, we conditionally deleted fibronectin in the osteoblasts and in the liver using the cre‐loxP system. We also used mice with mutated fibronectin and conditionally deleted β1‐integrin in osteoblasts to identify the receptor involved in fibronectin effects on osteoblasts. Conditional deletion of fibronectin in the differentiating osteoblasts [using the 2.3 kb collagen‐α1(I) promoter] failed to show a decrease in fibronectin amount in the bone matrix despite evidence of successful deletion. Using these mice we established that osteoblast‐derived fibronectin solely affects osteoblast function. This effect was not mediated by integrins that bind to the RGD motif. Conditional deletion of fibronectin in the liver showed a marked decrease in fibronectin content in the matrix associated with decreased mineral‐to‐matrix ratio and changed biomechanical properties but had no effect on osteoblasts or osteoclasts. In conclusion, osteoblast fibronectin affects osteoblasts function. This does not seem to be mediated by the RGD motif on fibronectin. In contrast, liver‐derived fibronectin affects bone matrix properties without affecting osteoblast or osteoclast function. A novel role for liver‐derived circulating fibronectin thus was defined and delineated from that of locally produced fibronectin.

The effect of pre-reduction of cytochrome b-563 on the electron-transfer reactions of the cytochrome bf complex in higher plant chloroplasts

Abstract An investigation has been made of the effects of pre-reduction of cytochrome b -563 on electron transfers through the cytochrome bf complex. It has been found that in a system in which anthraquinone-2-sulphonate or anthraquinone-2,6-disulphonate is used as redox buffer, a lipid-soluble mediator must also be present to allow sufficiently rapid equilibration of cytochrome b -563 with the ambient potential. We have found that 1 μM benzyl viologen gives full equilibration of cytochrome b -563 in less than 30 s, while minimizing the side reactions that have been observed with alternative mediators. Pre-reduction of cytochrome b -563 did not prevent turnover of site o (quinol-oxidising site of the cytochrome bc complex), even with fast repetitive flash activation. The site o reaction was accompanied by rapid, 2-nonyl-4-hydroxyquinoline N -oxide-sensitive oxidation of cytochrome b , and by a slow carotenoid bandshift. These results are discussed in conjunction with related results from the cytochrome bc 1 complex; Q-cycle models are considered in which the semiquinone at site o either can reduce an oxidant other than cytochrome b -563, or can migrate to site r (quinone-reducing site of the cytochrome bc complex). Of these possibilities, only the migration of the neutral semiquinone, QH, to site r is compatible with all of the data from the cytochrome bf and bc 1 complexes. Such a scheme would not be compatible with the semiquinone cycle proposed by Wikstrom and Krab ((1986) J. Bioenerg. Biomembr. 18, 181–193).

Toxic and non-toxic aggregates from the SBMA and normal forms of androgen receptor have distinct oligomeric structures

Hormone-dependent aggregation of the androgen receptor (AR) with a polyglutamine (polyQ) stretch amplification (>38) is considered to be the causative agent of the neurodegenerative disorder spinal and bulbar muscular atrophy (SBMA), consistent with related neurodegenerative diseases involving polyQ-extended proteins. In spite of the widespread acceptance of this common causal hypothesis, little attention has been paid to its apparent incompatibility with the observation of AR aggregation in healthy individuals with no polyQ stretch amplification. Here we used atomic force microscopy (AFM) to characterize sub-micrometer scale aggregates of the wild-type (22 glutamines) and the SBMA form (65 glutamines), as well as a polyQ deletion mutant (1 glutamine) and a variant with a normal length polyQ stretch but with a serine to alanine double mutation elsewhere in the protein. We used a baculovirus-insect cell expression system to produce full-length proteins for these structural analyses. We related the AFM findings to cytotoxicity as measured by expression of the receptors in Drosophila motoneurons or in neuronal cells in culture. We found that the pathogenic AR mutants formed oligomeric fibrils up to 300-600nm in length. These were clearly different from annular oligomers 120-180nm in diameter formed by the nonpathogenic receptors. We could also show that melatonin, which is known to ameliorate the pathological phenotype in the fly model, caused polyQ-extended AR to form annular oligomers. Further comparative investigation of these reproducibly distinct toxic and non-toxic oligomers could advance our understanding of the molecular basis of the polyQ pathologies.

Terahertz Radiation at ANKA, the New Synchrotron Light Source in Karlsruhe

ANKA is a new synchrotron light source atthe Karlsruhe Research Center in southwestGermany. The acronym stands for Ångstrøm Source Karlsruhe.The ANKA-IR beamline provides a highbrilliance infrared beam through the near,mid and far-infrared range. Thefar-infrared range is of particularinterest, since at frequencies lower thanaround 200 cm-1 (6 THz) synchrotronlight begins to outperform conventionalthermal sources in terms of total intensityas well as brilliance. The extraction ofthe entire flux is a challenge in the THzrange, since the natural verticaldivergence of synchrotron radiationincreases with wavelength and the openingangle for collection is limited by designconstraints. At ANKA-IR, this problem issolved by the collection of radiationemitted from a bending magnet edge source,which has a much smaller verticaldivergence than conventional synchrotronradiation emitted from the constantmagnetic field region within the dipolemagnet. Edge radiation at ANKA permits theextraction of the entire infrared flux downto around 100 cm-1 (3 THz) while withconventional synchrotron radiation thiswould only be the case for frequencies downto 2500 cm-1. ANKA-IR provides usableintensity down to 4 cm-1 (120 GHz).