Folding kinetics and structure of OEP16

The chloroplast outer membrane contains different, specialized pores that are involved in highly specific traffic processes from the cytosol into the chloroplast and vice versa. One representative member of these channels is the outer envelope protein 16 (OEP16), a cation-selective high conductance channel with high selectivity for amino acids. Here we study the mechanism and kinetics of the folding of this membrane protein by fluorescence and circular dichroism spectroscopy, using deletion mutants of the two single tryptophanes Trp-77-->Phe-77 and Trp-100-->Phe-100. In addition, the wild-type spectra were deconvoluted, depicting the individual contributions from each of the two tryptophan residues. The results show that both tryptophan residues are located in a completely different environment. The Trp-77 is deeply buried in the hydrophobic part of the protein, whereas the Trp-100 is partially solvent exposed. These results were further confirmed by studies of fluorescence quenching with I(-). The kinetics of the protein folding are studied by stopped flow fluorescence and circular dichroism measurements. The folding process depends highly on the detergent concentration and can be divided into an ultrafast phase (k > 1000 s(-1)), a fast phase (200-800 s(-1)), and a slow phase (25-70 s(-1)). The slow phase is absent in the W100F mutant. Secondary structure analysis and comparision with closely related proteins led to a new model of the structure of OEP16, suggesting that the protein is, in contrast to most other outer membrane proteins studied so far, purely alpha-helical, consisting of four transmembrane helices. Trp-77 is located in helix II, whereas the Trp-100 is located in the loop between helices II and III, close to the interface to helix III. We suggest that the first, very fast process corresponds to the formation of the helices, whereas the insertion of the helices into the detergent micelle and the correct folding of the II-III loop may be the later, rate-limiting steps of the folding process.     

Jointly They Edit: Examining the Impact of Community Identification on Political Interaction in Wikipedia

Background

In their 2005 study, Adamic and Glance coined the memorable phrase ‘divided they blog’, referring to a trend of cyberbalkanization in the political blogosphere, with liberal and conservative blogs tending to link to other blogs with a similar political slant, and not to one another. As political discussion and activity increasingly moves online, the power of framing political discourses is shifting from mass media to social media.

Methodology/Principal Findings

Continued examination of political interactions online is critical, and we extend this line of research by examining the activities of political users within the Wikipedia community. First, we examined how users in Wikipedia choose to display their political affiliation. Next, we analyzed the patterns of cross-party interaction and community participation among those users proclaiming a political affiliation. In contrast to previous analyses of other social media, we did not find strong trends indicating a preference to interact with members of the same political party within the Wikipedia community.

Conclusions/Significance

Our results indicate that users who proclaim their political affiliation within the community tend to proclaim their identity as a ‘Wikipedian’ even more loudly. It seems that the shared identity of ‘being Wikipedian’ may be strong enough to triumph over other potentially divisive facets of personal identity, such as political affiliation.     

A monomeric membrane peptide that lives in three worlds: in solution, attached to, and inserted across lipid bilayers

The membrane peptide pH (low) insertion peptide (pHLIP) lives in three worlds, being soluble in aqueous solution at pH 7.4, binding to the surface of lipid bilayers, and inserting as a transbilayer helix at low pH. With low pH driving the process, pHLIP can translocate cargo molecules attached to its C-terminus via a disulfide and release them in the cytoplasm of a cell. Here we examine a key aspect of the mechanism, showing that pHLIP is monomeric in each of its three major states: soluble in water near neutral pH (state I), bound to the surface of a membrane near neutral pH (state II), and inserted across the membrane as an alpha-helix at low pH (state III). The peptide does not induce fusion or membrane leakage. The unique properties of pHLIP made it attractive for the biophysical investigation of membrane protein folding in vitro and for the development of a novel class of delivery peptides for the transport of therapeutic and diagnostic agents to acidic tissue sites associated with various pathological processes in vivo.     

SNAP predicts effect of mutations on protein function

Many non-synonymous single nucleotide polymorphisms (nsSNPs) in humans are suspected to impact protein function. Here, we present a publicly available server implementation of the method SNAP (screening for non-acceptable polymorphisms) that predicts the functional effects of single amino acid substitutions. SNAP identifies over 80% of the non-neutral mutations at 77% accuracy and over 76% of the neutral mutations at 80% accuracy at its default threshold. Each prediction is associated with a reliability index that correlates with accuracy and thereby enables experimentalists to zoom into the most promising predictions.     

Differential modulation of the lipoxygenase cascade during typical and latent Pectobacterium atrosepticum infections

Background and AimsPlant diseases caused by Pectobacterium atrosepticum are often accompanied by extensive rot symptoms. In addition, these bacteria are able to interact with host plants without causing disease for long periods, even throughout several host plant generations. There is, to date, no information on the comparative physiology/biochemistry of symptomatic and asymptomatic plant–P. atrosepticum interactions. Typical (symptomatic) P. atrosepticum infections are associated with the induction of plant responses mediated by jasmonates, which are one of the products of the lipoxygenase cascade that gives origin to many other oxylipins with physiological activities. In this study, we compared the functioning of the lipoxygenase cascade following typical and latent (asymptomatic) infections to gain better insight into the physiological basis of the asymptomatic and antagonistic coexistence of plants and pectobacteria.MethodsTobacco plants were mock-inoculated (control) or infected with the wild type P. atrosepticum (typical infection) or its coronafacic acid-deficient mutant (latent infection). The expression levels of the target lipoxygenase cascade-related genes were assessed by Illumina RNA sequencing. Oxylipin profiles were analysed by GC-MS. With the aim of revising the incorrect annotation of one of the target genes, its open reading frame was cloned to obtain the recombinant protein, which was further purified and characterized using biochemical approaches.Key ResultsThe obtained data demonstrate that when compared to the typical infection, latent asymptomatic P. atrosepticum infection is associated with (and possibly maintained due to) decreased levels of 9-lipoxygenase branch products and jasmonic acid and increased level of cis-12-oxo-10,15-phytodienoic acid. The formation of 9-oxononanoic acid and epoxyalcohols in tobacco plants was based on the identification of the first tobacco hydroperoxide lyase (HPL) with additional epoxyalcohol synthase (EAS) activity.ConclusionsOur results contribute to the hypothesis of the oxylipin signature, indicating that different types of plant interactions with a particular pathogen are characterized by the different oxylipin profiles of the host plant. In addition, the tobacco LOC107825278 gene was demonstrated to encode an NtHPL (CYP74C43) enzyme yielding volatile aldehydes and aldoacids (HPL products) as well as oxiranyl carbinols (EAS products).     

Improved Open‐ Circuit Voltage in ZnO–PbSe Quantum Dot Solar Cells by Understanding and Reducing Losses Arising from the ZnO Conduction Band Tail

Colloidal quantum dot solar cells (CQDSCs) are attracting growing attention owing to significant improvements in efficiency. However, even the best depleted‐heterojunction CQDSCs currently display open‐circuit voltages (V_OCs) at least 0.5 V below the voltage corresponding to the bandgap. We find that the tail of states in the conduction band of the metal oxide layer can limit the achievable device efficiency. By continuously tuning the zinc oxide conduction band position via magnesium doping, we probe this critical loss pathway in ZnO–PbSe CQDSCs and optimize the energetic position of the tail of states, thereby increasing both the V_OC (from 408 mV to 608 mV) and the device efficiency.     

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co‐localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site‐specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild‐type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild‐type Arabidopsis. Thus, we conclude that the PM‐associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity‐dependent manner, rather than its subcellular translocation.     

Improved Performance and Stability of Inverted Organic Solar Cells with Sol–Gel Processed,Amorphous Mixed Metal Oxide Electron Extraction Layers Comprising Alkaline Earth Metals

Stability of organic photovoltaic devices (OPVs) is a limiting factor for their commercialization and still remains a major challenge whilst power conversion efficiencies are now approaching minimum requirements. The inverted organic solar cell (iOSC) architecture shows promising potential for improving significantly the cell's working lifetime. However, when solution processed ZnO is used as electron extraction layer, an undesirable light‐soaking step is commonly required before the device reaches a non‐permanent maximum performance. This work investigates the use of Sr and Ba doped ZnO films, ZnSrO and ZnBaO, formed by sol‐gel deposition using molecular alkoxide precursor solutions, as electron extraction layers in a model iOSCs with poly [3‐hexylthiophene] (P3HT): [6, 6]‐phenyl C60 butyl acid methyl ester (PCBM) as the active layer. We show that using these ternary oxides the light‐soaking step can be circumvented by preventing a dipole forming between the oxide and the active organic layer as supported by electroabsorption spectroscopy measurements of the device built‐in field. It is suggested that Sr or Ba doping results in suppression/reduction of the oxygen adsorption at mobile oxygen vacancy sites on the metal oxide surface. Like in thin film transistor (TFT) applications, where materials like InGaZnO are rapidly becoming an important technology, the use of amorphous, mixed metal oxides allows improving the performance and stability of interfacial charge extraction layers for organic solar cells.