Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase

14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.     

Hermit crabs and their symbionts: Reactions to artificially induced anoxia on a sublittoral sediment bottom

Hermit crabs play an important role in the Northern Adriatic Sea due to their abundance, wide range of symbionts, and function in structuring the benthic community. Small-scale (0.25?m(2)) hypoxia and anoxia were experimentally generated on a sublittoral soft bottom in 24?m depth in the Gulf of Trieste. This approach successfully simulates the seasonal low dissolved oxygen (DO) events here and enabled studying the behaviour and mortality of the hermit crab Paguristes eremita. The crabs exhibited a sequence of predictable stress responses and ultimately mortality, which was correlated with five oxygen thresholds. Among the crustaceans, which are a sensitive group to oxygen depletion, P. eremita is relatively tolerant. Initially, at mild hypoxia (2.0 to 1.0?ml?l(-?1) DO), hermit crabs showed avoidance by moving onto better oxygenated, elevated substrata. This was accompanied by a series of responses including decreased locomotory activity, increased body movements and extension from the shell. During a moribund phase at severe hypoxia (0.5 to 0.01?ml?l(-?1) DO), crabs were mostly immobile in overturned shells and body movements decreased. Anoxia triggered emergence from the shell, with a brief locomotion spurt of shell-less crabs. The activity pattern of normally day-active crabs was altered during hypoxia and anoxia. Atypical interspecific interactions occurred: the crab Pisidia longimana increasingly aggregated on hermit crab shells, and a hermit crab used the emerged infaunal sea urchin Schizaster canaliferus as an elevated substrate. Response patterns varied somewhat according to shell size or symbiont type (the sponge Suberites domuncula). Mortality occurred after extended anoxia (~?1.5?d) and increased hydrogen sulphide levels (H(2)S ~?128?μmol). The relative tolerance of crabs and certain symbionts (e.g. the sea anemone Calliactis parasitica) - as potential survivors and recolonizers of affected areas - may influence and promote community recovery after oxygen crises.     

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T‐DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, α‐tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus‐end marker GFP–EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell‐to‐cell movement of TMV encoding GFP‐tagged movement protein (MP‐GFP) is reduced in ATER2 accompanied by a reduced association of MP‐GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.     

Efficient identification of near‐native conformations in ab initio protein structure prediction using structural profiles

One of the major bottlenecks in many ab initio protein structure prediction methods is currently the selection of a small number of candidate structures for high‐resolution refinement from large sets of low‐resolution decoys. This step often includes a scoring by low‐resolution energy functions and a clustering of conformations by their pairwise root mean square deviations (RMSDs). As an efficient selection is crucial to reduce the overall computational cost of the predictions, any improvement in this direction can increase the overall performance of the predictions and the range of protein structures that can be predicted. We show here that the use of structural profiles, which can be predicted with good accuracy from the amino acid sequences of proteins, provides an efficient means to identify good candidate structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

In vitro reconstitution of fibrillar collagen type I assemblies at reactive polymer surfaces

The reconstitution of fibrillar collagen and its assemblies with heparin and hyaluronic acid was studied in vitro. Fibril formation kinetics were analyzed by turbidity and depletion measurements in solutions containing varied concentrations of collagen and glycosaminoglycans. Fibril-forming collagen solutions were further applied for the coating of planar substrates which had been modified with alternating maleic anhydride copolymer films before. The immobilized collagen assemblies were characterized with respect to the deposited amount of protein using ellipsometry and acidic hydrolysis/HPLC-based amino acid analysis, respectively. AFM, SEM, and cLSM were utilized to gain information on structural features and patterns formed by surface-attached fibrils depending on the initial solution concentrations of collagen. The results revealed that the addition of heparin and hyaluronic acid affected both the fibril dimensions and the meshwork characteristics of the surface-bound fibrils.     

Microbial cycling of iron and sulfur in sediments of acidic and pH-neutral mining lakes in Lusatia (Brandenburg, Germany)

A vast number of lakes developed in the abandoned opencast lignite mines of Lusatia (East Germany) contain acidic waters (mmolSm^–2a^–). Potential Fe(III) reduction measured by the accumulation of Fe(II) during anoxic incubation yielded similar rates in both types of sediments, however, the responses towards the supplementation of Fe(III) and organic carbon were different. Sulfate reduction rates estimated with ³⁵S-radiotracer were much lower in the slightly acidic sediment than in the pH-neutral sediment (156 v.s. 738mmolSO₄ ^2–m^–2a^–1). However, sulfate reduction rates were increased by the addition of organic carbon. Severe limitation of sulfate-reducing bacteria under acidic conditions was also reflected by low most probable numbers (MPN). High MPN of acidophilic iron- and sulfur-oxidizing bacteria in acidic sediments indicated a high reoxidation potential. The results show that potentials for reductive processes are present in acidic sediments and that these are determined mainly by the availability of oxidants and organic matter.     

Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum

The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex.