Long-range structural and dynamical changes induced by cofactor binding in DNA methyltransferase M.HhaI

The bacterial DNA cytosine methyltransferase M.HhaI sequence-specifically modifies DNA in an S-adenosylmethionine dependent reaction. The enzyme stabilizes the target cytosine (GCGC) into an extrahelical position, with a concomitant large movement of an active site loop involving residues 80-99. We used multidimensional, transverse relaxation-optimized NMR experiments to assign nearly 80% of all residues in the cofactor-bound enzyme form, providing a basis for detailed structural and dynamical characterization. We examined details of the previously unknown effects of the cofactor binding with M.HhaI in solution. Addition of the cofactor results in numerous structural changes throughout the protein, including those decorating the cofactor binding site, and distal residues more than 30 A away. The active site loop is involved in motions both on a picosecond to nanosecond time scale and on a microsecond to millisecond time scale and is not significantly affected by cofactor binding except for a few N-terminal residues. The cofactor also affects residues near the DNA binding cleft, suggesting a role for the cofactor in regulating DNA interactions. The allosteric properties we observed appear to be closely related to the significant amount of dynamics and dynamical changes in response to ligand binding detected in the protein.     

Differential regulation of protein phosphatase-1(I) by neurabin

Neurabin is a brain-specific actin and protein phosphatase-1 (PP-1) binding protein that inhibits the purified catalytic subunit of protein phosphatase-1 (PP-1(C)). However, endogenous PP-1 exists primarily as multimeric complexes of PP-1(C) bound to various regulatory proteins that determine its activity, substrate specificity, subcellular localization and function. The major form of endogenous PP-1 in brain is protein phosphatase-1(I) (PP-1(I)), a Mg(2+)/ATP-dependent form of PP-1 that consists of PP-1(C), the inhibitor-2 regulatory subunit, an activating protein kinase and other unidentified proteins. We have identified four PP-1(I) holoenzyme fractions (PP-1(IA), PP-1(IB), PP-1(IC), and PP-1(ID)) in freshly harvested pig brain separable by poly-L-lysine chromatography. Purified recombinant neurabin (amino acid residues 1-485) inhibited PP-1(IB) (IC(50)=1.1 microM), PP-1(IC) (IC(50)=0.1 microM), and PP-1(ID) (IC(50)=0.2 microM), but activated PP-1(IA) by up to threefold (EC(50)=40 nM). The PP-1(IA) activation domain was localized to neurabin(1-210). Our results indicate a novel mechanism of PP-1 regulation by neurabin as both an inhibitor and an activator of distinct forms of PP-1(I) in brain.     

Directed Mutation of the Rubisco Large Subunit of Tobacco Influences Photorespiration and Growth

The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO2 enrichment. In 0.3% (v/v) CO2, the mutant and wild-type plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO2-fixing rate and its ability to distinguish between CO2 and O2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO2, O2, and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O2 concentrations. Consistent with the reduced CO2 fixation rate at saturating CO2, the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.     

Forecasted range shifts of arid-land fishes in response to climate change

Climate change is poised to alter the distributional limits, center, and size of many species. Traits may influence different aspects of range shifts, with trophic generality facilitating shifts at the leading edge, and greater thermal tolerance limiting contractions at the trailing edge. The generality of relationships between traits and range shifts remains ambiguous however, especially for imperiled fishes residing in xeric riverscapes. Our objectives were to quantify contemporary fish distributions in the Lower Colorado River Basin, forecast climate change by 2085 using two general circulation models, and quantify shifts in the limits, center, and size of fish elevational ranges according to fish traits. We examined relationships among traits and range shift metrics either singly using univariate linear modeling or combined with multivariate redundancy analysis. We found that trophic and dispersal traits were associated with shifts at the leading and trailing edges, respectively, although projected range shifts were largely unexplained by traits. As expected, piscivores and omnivores with broader diets shifted upslope most at the leading edge while more specialized invertivores exhibited minimal changes. Fishes that were more mobile shifted upslope most at the trailing edge, defying predictions. No traits explained changes in range center or size. Finally, current preference explained multivariate range shifts, as fishes with faster current preferences exhibited smaller multivariate changes. Although range shifts were largely unexplained by traits, more specialized invertivorous fishes with lower dispersal propensity or greater current preference may require the greatest conservation efforts because of their limited capacity to shift ranges under climate change.     

Influence of experimental,environmental, and geographic factors on nutrient‐diffusing substrate experiments in running waters

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Nectar discovery speeds and multimodal displays: assessing nectar search times in bees with radiating and non-radiating guides

Floral displays are often composed of areas of contrasting stimuli which flower visitors use as guides, increasing both foraging efficiency and the likelihood of pollen transfer. Many aspects of how these displays benefit foraging efficiency are still unexplored, particularly those surrounding multimodal signals and the spatial arrangement of the display components. We compare the nectar discovery times of forager bumblebees (Bombus terrestris) when presented with artificial flowers with unimodal or compound displays of visual and/or olfactory stimuli, positioned in either radiating or non-radiating arrangements. We found that the addition of individual display components from either modality reduces nectar discovery time but there was no time benefit to bimodal displays over unimodal displays or any benefit to radiating stimuli arrangements over non-radiating arrangements. However, preference tests revealed a time advantage to radiating unimodal visual patterns over non-radiating unimodal visual patterns when both types were displayed simultaneously. These results suggest that the benefits of multimodal stimuli arrangements to pollinators are unrelated to benefits in nectar discovery time. Our results also suggest that spatial patterns of scent can be used as nectar guides and can reduce nectar discovery times without the aid of visual stimuli.     

Are mesophotic coral ecosystems distinct communities and can they serve as refugia for shallow reefs?

We analyzed an extensive dataset of over 9000 benthic and suprabenthic species found throughout the Gulf of Mexico (GoMx) to assess whether mesophotic coral ecosystems represent distinct assemblages and evaluate their potential to serve as refugia for shallow reef communities. We assessed community structure of the overall benthic community from 0 to 300 m via non-metric multidimensional scaling (NMDS) of species presence across depth bands. We used the Jaccard index of similarity to calculate the proportion of shared species between adjacent depth bands, measure species turnover with depth, and assess taxonomic overlap between shallow reefs versus progressively deeper depth bands. NMDS ordinations showed that the traditionally defined mesophotic range (30–150 m) as a whole is not a distinct community. In contrast, taxonomically distinct communities, determined by hierarchical clustering, were found at 0–70, 60–120, 110–200, and 190–300 m. Clustering highlighted an important separation in the benthic community at ~60 m, which was especially important for actinopterygian fishes. Species turnover between adjacent depths decreased with depth for all taxa combined and individual taxa, with peaks at ~60, 90–120, and 190–200 m. Fishes showed lower turnover from shallow to upper mesophotic depths (0–50 m) than all taxa combined, a substantial peak at 60 m, followed by a precipitous and continued decline in turnover thereafter. Taxonomic overlap between shallow (0–20 m) and progressively deeper zones declined steadily with depth in all taxa and individual taxa, suggesting that mid- and lower mesophotic habitats have less (but not inconsequential) potential to serve as refugia (60–150 m, 15–25% overlap with shallow habitats) than upper mesophotic zones (30–60 m, 30–45% overlap with shallow habitats) for all taxa combined. We conclude that the traditional mesophotic zone is home to three ecological communities in the GoMx, one that is confluent with shallow reefs, a distinct mesophotic assemblage spanning 60–120 m, and a third that extends onto the outer continental shelf.     

A Low Resistance Calcium/Reduced Titania Passivated Contact for High Efficiency Crystalline Silicon Solar Cells

Recent advances in the efficiency of crystalline silicon (c‐Si) solar cells have come through the implementation of passivated contacts that simultaneously reduce recombination and resistive losses within the contact structure. In this contribution, low resistivity passivated contacts are demonstrated based on reduced titania (TiO_x) contacted with the low work function metal, calcium (Ca). By using Ca as the overlying metal in the contact structure we are able to achieve a reduction in the contact resistivity of TiO_x passivated contacts of up to two orders of magnitude compared to previously reported data on Al/TiO_x contacts, allowing for the application of the Ca/TiO_x contact to n‐type c‐Si solar cells with partial rear contacts. Implementing this contact structure on the cell level results in a power conversion efficiency of 21.8% where the Ca/TiO_x contact comprises only ≈6% of the rear surface of the solar cell, an increase of 1.5% absolute compared to a similar device fabricated without the TiO_x interlayer.     

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.