Sequential nearest-neighbor effects on computed <Superscript>13</Superscript>C<Superscript>α</Superscript> chemical shifts

To evaluate sequential nearest-neighbor effects on quantum-chemical calculations of ¹³C^α chemical shifts, we selected the structure of the nucleic acid binding (NAB) protein from the SARS coronavirus determined by NMR in solution (PDB id 2K87). NAB is a 116-residue α/β protein, which contains 9 prolines and has 50% of its residues located in loops and turns. Overall, the results presented here show that sizeable nearest-neighbor effects are seen only for residues preceding proline, where Pro introduces an overestimation, on average, of 1.73 ppm in the computed ¹³C^α chemical shifts. A new ensemble of 20 conformers representing the NMR structure of the NAB, which was calculated with an input containing backbone torsion angle constraints derived from the theoretical ¹³C^α chemical shifts as supplementary data to the NOE distance constraints, exhibits very similar topology and comparable agreement with the NOE constraints as the published NMR structure. However, the two structures differ in the patterns of differences between observed and computed ¹³C^α chemical shifts, Δ _ca,i , for the individual residues along the sequence. This indicates that the Δ _ca,i -values for the NAB protein are primarily a consequence of the limited sampling by the bundles of 20 conformers used, as in common practice, to represent the two NMR structures, rather than of local flaws in the structures.     

Predicting <Superscript>13</Superscript>C<Superscript>α</Superscript> chemical shifts for validation of protein structures

The ¹³C^α chemical shifts for 16,299 residues from 213 conformations of four proteins (experimentally determined by X-ray crystallography and Nuclear Magnetic Resonance methods) were computed by using a combination of approaches that includes, but is not limited to, the use of density functional theory. Initially, a validation test of this methodology was carried out by a detailed examination of the correlation between computed and observed ¹³C^α chemical shifts of 10,564 (of the 16,299) residues from 139 conformations of the human protein ubiquitin. The results of this validation test on ubiquitin show agreement with conclusions derived from computation of the chemical shifts at the ab initio Hartree–Fock level. Further, application of this methodology to 5,735 residues from 74 conformations of the three remaining proteins that differ in their number of amino acid residues, sequence and three-dimensional structure, together with a new scoring function, namely the conformationally averaged root-mean-square-deviation, enables us to: (a) offer a criterion for an accurate assessment of the quality of NMR-derived protein conformations; (b) examine whether X-ray or NMR-solved structures are better representations of the observed ¹³C^α chemical shifts in solution; (c) provide evidence indicating that the proposed methodology is more accurate than automated predictors for validation of protein structures; (d) shed light as to whether the agreement between computed and observed ¹³C^α chemical shifts is influenced by the identity of an amino acid residue or its location in the sequence; and (e) provide evidence confirming the presence of dynamics for proteins in solution, and hence showing that an ensemble of conformations is a better representation of the structure in solution than any single conformation. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Phenotypic shifts caused by predation: selection or life-history shifts?

Predators can impose both selection and life-history shifts in prey populations. Because both processes may affect phenotypic distributions, the estimates of selection differentials may be biased. We carried out two field experiments to disentangle these separate effects. We studied whether dragonfly predation by Aeshna cyanea changes the distributions in body size and lamellae morphology in the damselfly Lestes sponsa. Damselflies have caudal lamellae which are used in escapes by swimming. In a first experiment, we manipulated predator presence (No Aeshna, Encaged Aeshna or Free-ranging Aeshna) and stopped the experiment when all larvae had moulted once. In a second experiment, larvae were confronted with a Free-ranging Aeshna but collected before moulting, and survivors were compared with a control sample taken at the start of the experiment. The presence of Aeshna largely reduced the survival probabilities of the Lestes larvae at a very similar rate in both experiments. Daily survival probabilities did not differ between the No Aeshna and Encaged Aeshna treatments. In the Free-ranging Aeshna treatment of the first experiment, size was reduced compared to the other two treatments, creating a significant apparent selection differential. This was probably mainly due to predator-induced reduced growth because in the second experiment, where growth effects were excluded, size of the survivors did not differ from the control sample. In both experiments there was a significant selection pressure for larger lamellae. Standardized directional selection differentials were similar in both experiments (0.57 and 0.28 phenotypic standard deviation units). No survival selection on lamellae shape was detected. These results are in agreement with previous findings that lamellae size, but not lamellae shape, enhances swimming performance and thereby predator escape in this species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Do species’ traits predict recent shifts at expanding range edges?

Although some organisms have moved to higher elevations and latitudes in response to recent climate change, there is little consensus regarding the capacity of different species to track rapid climate change via range shifts. Understanding species' abilities to shift ranges has important implications for assessing extinction risk and predicting future community structure. At an expanding front, colonization rates are determined jointly by rates of reproduction and dispersal. In addition, establishment of viable populations requires that individuals find suitable resources in novel habitats. Thus, species with greater dispersal ability, reproductive rate and ecological generalization should be more likely to expand into new regions under climate change. Here, we assess current evidence for the relationship between leading-edge range shifts and species' traits. We found expected relationships for several datasets, including diet breadth in North American Passeriformes and egg-laying habitat in British Odonata. However, models generally had low explanatory power. Thus, even statistically and biologically meaningful relationships are unlikely to be of predictive utility for conservation and management. Trait-based range shift forecasts face several challenges, including quantifying relevant natural history variation across large numbers of species and coupling these data with extrinsic factors such as habitat fragmentation and availability.     

Measuring <Superscript>13</Superscript>C<Superscript>β</Superscript> chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

A labeling scheme is introduced that facilitates the measurement of accurate ¹³C^β chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of ¹³C enrichment (30–40%) at C^β side-chain carbon positions for 15 of the amino acids with little ¹³C label at positions one bond removed (≈5%). A pair of samples are produced using [1-¹³C]-glucose/NaH¹²CO₃ or [2-¹³C]-glucose as carbon sources with isolated and enriched (>30%) ¹³C^β positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of ¹³C^β chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein–ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.     

Effects of side-chain orientation on the <Superscript>13</Superscript>C chemical shifts of antiparallel β-sheet model peptides

The dependence of the ¹³C chemical shift on side-chain orientation was investigated at the density functional level for a two-strand antiparallel β-sheet model peptide represented by the amino acid sequence Ac-(Ala)₃-X-(Ala)₁₂-NH₂ where X represents any of the 17 naturally occurring amino acids, i.e., not including alanine, glycine and proline. The dihedral angles adopted for the backbone were taken from, and fixed at, observed experimental values of an antiparallel β-sheet. We carried out a cluster analysis of the ensembles of conformations generated by considering the side-chain dihedral angles for each residue X as variables, and use them to compute the ¹³C chemical shifts at the density functional theory level. It is shown that the adoption of the locally-dense basis set approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 17 naturally occurring amino acids in an antiparallel β-sheet, there is (i) good agreement between computed and observed ¹³C^α and ¹³C^β chemical shifts, with correlation coefficients of 0.95 and 0.99, respectively; (ii) significant variability of the computed ¹³C^α and ¹³C^β chemical shifts as a function of χ¹ for all amino acid residues except Ser; and (iii) a smaller, although significant, dependence of the computed ¹³C^α chemical shifts on χ^ξ (with ξ ≥ 2) compared to χ¹ for eleven out of seventeen residues. Our results suggest that predicted ¹³C^α and ¹³C^β chemical shifts, based only on backbone (φ,ψ) dihedral angles from high-resolution X-ray structure data or from NMR-derived models, may differ significantly from those observed in solution if the dihedral-angle preferences for the side chains are not taken into account. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Hash: a program to accurately predict protein Hα shifts from neighboring backbone shifts

Chemical shifts provide not only peak identities for analyzing nuclear magnetic resonance (NMR) data, but also an important source of conformational information for studying protein structures. Current structural studies requiring H^α chemical shifts suffer from the following limitations. (1) For large proteins, the H^α chemical shifts can be difficult to assign using conventional NMR triple-resonance experiments, mainly due to the fast transverse relaxation rate of C^α that restricts the signal sensitivity. (2) Previous chemical shift prediction approaches either require homologous models with high sequence similarity or rely heavily on accurate backbone and side-chain structural coordinates. When neither sequence homologues nor structural coordinates are available, we must resort to other information to predict H^α chemical shifts. Predicting accurate H^α chemical shifts using other obtainable information, such as the chemical shifts of nearby backbone atoms (i.e., adjacent atoms in the sequence), can remedy the above dilemmas, and hence advance NMR-based structural studies of proteins. By specifically exploiting the dependencies on chemical shifts of nearby backbone atoms, we propose a novel machine learning algorithm, called Hash, to predict H^α chemical shifts. Hash combines a new fragment-based chemical shift search approach with a non-parametric regression model, called the generalized additive model, to effectively solve the prediction problem. We demonstrate that the chemical shifts of nearby backbone atoms provide a reliable source of information for predicting accurate H^α chemical shifts. Our testing results on different possible combinations of input data indicate that Hash has a wide rage of potential NMR applications in structural and biological studies of proteins.     

Are fish outside their usual ranges early indicators of climate‐driven range shifts?

Shifts in species ranges are a global phenomenon, well known to occur in response to a changing climate. New species arriving in an area may become pest species, modify ecosystem structure, or represent challenges or opportunities for fisheries and recreation. Early detection of range shifts and prompt implementation of any appropriate management strategies is therefore crucial. This study investigates whether ‘first sightings’ of marine species outside their normal ranges could provide an early warning of impending climate‐driven range shifts. We examine the relationships between first sightings and marine regions defined by patterns of local climate velocities (calculated on a 50‐year timescale), while also considering the distribution of observational effort (i.e. number of sampling days recorded with biological observations in global databases). The marine trajectory regions include climate ‘source’ regions (areas lacking connections to warmer areas), ‘corridor’ regions (areas where moving isotherms converge), and ‘sink’ regions (areas where isotherms locally disappear). Additionally, we investigate the latitudinal band in which first sightings were recorded, and species’ thermal affiliations. We found that first sightings are more likely to occur in climate sink and ‘divergent’ regions (areas where many rapid and diverging climate trajectories pass through) indicating a role of temperature in driving changes in marine species distributions. The majority of our fish first sightings appear to be tropical and subtropical species moving towards high latitudes, as would be expected in climate warming. Our results indicate that first sightings are likely related to longer‐term climatic processes, and therefore have potential use to indicate likely climate‐driven range shifts. The development of an approach to detect impending range shifts at an early stage will allow resource managers and researchers to better manage opportunities resulting from range‐shifting species before they potentially colonize.     

A <Superscript>13</Superscript>C-detected <Superscript>15</Superscript>N double-quantum NMR experiment to probe arginine side-chain guanidinium <Superscript>15</Superscript>N<Superscript>η</Superscript> chemical shifts

Arginine side-chains are often key for enzyme catalysis, protein–ligand and protein–protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine ¹³C^ζ-detected NMR experiment in which a double-quantum coherence involving the two ¹⁵N^η nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two ¹⁵N^η; this new approach is shown to eliminate the previously deleterious line broadenings of ¹⁵N^η resonances caused by the partially restricted rotation about the C^ζ–N^ε bond. Consequently, sharp and well-resolved ¹⁵N^η resonances can be observed. The utility of the presented method is demonstrated on the L99A mutant of the 19 kDa protein T4 lysozyme, where the measurement of small chemical shift perturbations, such as one-bond deuterium isotope shifts, of the arginine amine ¹⁵N^η nuclei becomes possible using the double-quantum experiment.     

Are ontogenetic shifts in foliar structure and resource acquisition spatially conditioned in tank‐bromeliads?

The phenotypic plasticity of plants has been explored as a function of either ontogeny (apparent plasticity) or environment (adaptive plasticity), although few studies have analyzed these factors together. In the present study, we take advantage of the dispersal of Aechmea mertensii bromeliads by Camponotus femoratus or Pachycondyla goeldii ants in shaded and sunny environments, respectively, to quantify ontogenetic changes in morphological, foliar, and functional traits, and to analyze ontogenetic and ant species effects on 14 traits. Most of the morphological (plant height, number of leaves), foliar (leaf thickness, leaf mass area, total water content, trichome density), and functional (leaf δ¹³C) traits differed as a function of ontogeny. Conversely, only leaf δ¹⁵N showed an adaptive phenotypic plasticity. On the other hand, plant width, tank width, longest leaf length, stomatal density, and leaf C concentration showed an adaptation to local environment with ontogeny. The exception was leaf N concentration, which showed no trend at all. Aechmea mertensii did not show an abrupt morphological modification such as in heteroblastic bromeliads, although it was characterized by strong, size‐related functional modifications for CO₂ acquisition. The adaptive phenotypic variation found between the two ant species indicates the spatially conditioned plasticity of A. mertensii in the context of insect‐assisted dispersal. However, ant‐mediated effects on phenotypic plasticity in A. mertensii are not obvious because ant species and light environment are confounding variables. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 299–312.     

Biometry of Upper Cretaceous (Cenomanian–Maastrichtian) coccoliths – a record of long-term stability and interspecies size shifts

Biometric measurements of Mesozoic coccoliths (coccolith length and width) have been used in short-term biostratigraphic, taxonomic and palaeoecologic studies, but until now, not over longer time scales. Here, we present a long time-series study (∼ 30 million years) for the Upper Cretaceous, which aims to identify broad trends in coccolith size and to understand the factors governing coccolith size change over long time scales. We have generated biometric data for the dominant Upper Cretaceous coccolith groups, Broinsonia/Arkhangelskiella, Prediscosphaera, Retecapsa and Watznaueria, from 36 Cenomanian–Maastrichtian (100.5–66 Ma) samples from Goban Spur in the northeast Atlantic (DSDP Site 549). These data show that the coccolith sizes within Prediscosphaera, Retecapsa and Watznaueria were relatively stable through the Late Cretaceous, with mean size variation less than 0.7 μm. Within the Broinsonia/Arkhangelskiella group there was more pronounced variation, with a mean size increase from ∼ 6 μm in the Cenomanian to ∼ 10 μm in the Campanian. This significant change in mean size was largely driven by evolutionary turnover (species origination and extinctions), and, in particular, the appearance of larger species/subspecies (Broinsonia parca parca, Broinsonia parca constricta, Arkhangelskiella cymbiformis) in the early Campanian, replacing smaller species, such as Broinsonia signata and Broinsonia enormis. Shorter-term size fluctuations within Broinsonia/Arkhangelskiella, observed across the Late Cenomanian–Turonian and Late Campanian–Maastrichtian intervals, may, however, reflect changing palaeoenvironmental conditions, such as sea surface temperature and nutrient availability.     

Within‐season increase in parental investment in a long‐lived bird species: investment shifts to maximize successful reproduction?

In nest‐building species predation of nest contents is a main cause of reproductive failure and parents have to trade off reproductive investment against antipredatory behaviours. While this trade‐off is modified by lifespan (short‐lived species prioritize current reproduction; long‐lived species prioritize future reproduction), it may vary within a breeding season, but this idea has only been tested in short‐lived species. Yet, life history theory does not make any prediction how long‐lived species should trade off current against future reproductive investment within a season. Here, we investigated this trade‐off through predator‐exposure experiments in a long‐lived bird species, the brown thornbill. We exposed breeding pairs that had no prior within‐season reproductive success to the models of a nest predator and a predator of adults during their first or second breeding attempt. Overall, parents reduced their feeding rate in the presence of a predator, but parents feeding second broods were more risk sensitive and almost ceased feeding when exposed to both types of predators. However, during second breeding attempts, parents had larger clutches and a higher feeding rate in the absence of predators than during first breeding attempts and approached both types of predators closer when mobbing. Our results suggest that the trade‐off between reproductive investment and risk‐taking can change in a long‐lived species within a breeding season depending on both prior nest predation and renesting opportunities. These patterns correspond to those in short‐lived species, raising the question of whether a within‐season shift in reproductive investment trade‐offs is independent of lifespan.     

Altering the substrate specificity site of <Emphasis Type="Italic">Aspergillus niger</Emphasis> PhyB shifts the pH optimum to pH 3.2

Phytases are of biotechnological importance as animal feed additives for their ability to catalyze the hydrolysis of phosphate from phytate for absorption by simple-stomached animals, and to reduce their fecal phosphorus excretion. Aspergillus niger PhyB has high catalytic activity at low pHs around 2.5, but has little activity at the commonly observed gastric pH of young animals (3.0–3.5). Our objective was to determine if the pH optima of PhyB could be broadened to a more characteristic pH range in the stomach of young animals through site-directed mutagenesis. We created two mutants, E272K and E272Q, each with a single amino acid substitution of the same residue in the substrate specificity site. Mutants were designed to replace an acidic amino acid, with either a neutral amino acid (E272Q) or basic amino acid (E272K), and were overexpressed in the yeast Pichia pastoris. While the wild-type (WT) pH optimum was 2.5, mutant E272K shifted to a new optimum of pH 3.2. E272K had a concomitant reduction in K _m of 36-fold at pH 2.5 and 6-fold at pH 3.2 compared to the WT. Our results indicate that the pH optimum of PhyB can be altered to match the stomach pH, along with an improved substrate affinity.     

Protein–protein HADDocking using exclusively pseudocontact shifts

In order to enhance the structure determination process of macromolecular assemblies by NMR, we have implemented long-range pseudocontact shift (PCS) restraints into the data-driven protein docking package HADDOCK. We demonstrate the efficiency of the method on a synthetic, yet realistic case based on the lanthanide-labeled N-terminal ε domain of the E. coli DNA polymerase III (ε186) in complex with the HOT domain. Docking from the bound form of the two partners is swiftly executed (interface RMSDs < 1 Å) even with addition of very large amount of noise, while the conformational changes of the free form still present some challenges (interface RMSDs in a 3.1–3.9 Å range for the ten lowest energy complexes). Finally, using exclusively PCS as experimental information, we determine the structure of ε186 in complex with the HOT-homologue θ subunit of the E. coli DNA polymerase III.     

Paradigm shifts in pathophysiology and management of atrial fibrillation—a tale of the RACE trials in the Netherlands

In the past 20 years the Netherlands-based RACE trials have investigated important concepts in clinical atrial fibrillation (AF). Their scope ranged from rhythm versus rate control to early or delayed cardioversion and also included early comprehensive management of AF in two trials, one focusing on early ‘upstream therapy’ and risk factor management and the other on integrated chronic nurse-led care. Studies were mostly triggered by simple clinical observations including futility of electrical cardioversion in persistent AF; many patients with permanent AF tolerating day-after-day ‘uncontrolled’ resting heart rates of up till 110 beats/min; patients being threatened more by vascular risks than AF itself; and insufficient guideline-based treatments for AF. Also the observation that recent-onset atrial fibrillation generally converts spontaneously, obviating cardioversion, triggered one of the studies. The RACE trials shifted a number of paradigms and by that could change the AF guidelines. The initial ‘shock-and-forget’ attitude made place for increased attention for anticoagulation, and in turn, broader vascular risks were recognised. In a nutshell, the adage eventually became: ‘look beyond the ECG, treat the patient’.

     

Palaeoenvironment shifts during last ~ 500 years and eutrophic evolution of the Wular Lake,Kashmir Valley,India

Limnology - Multi-proxy analysis of lake sediments provides high-resolution and reliable palaeoclimate records. The present study aims to investigate the palaeoenvironmental changes and eutrophic...     

Modified dispersal‐related traits in disjunct populations of bird‐dispersed Frangula alnus (Rhamnaceae): a result of its Quaternary distribution shifts?

Many European tree species survived Pleistocene glaciations in Mediterranean refugia and rapidly recolonized temperate Europe afterwards. Inter‐ and postglacial migration processes are assumed to have catalized evolutionary optimizations of dispersal‐related traits, but up to now empirical evidence is lacking in vertebrate‐dispersed plants. We investigated if south Iberian glacial relict and central European “colonizer” populations of the bird‐dispersed tree Frangula alnus have experienced differentiations of dispersal‐related traits which increase the mobility of northern populations. A comparison of lifetime reproductive strategy, disperser guilds, ripening phenology, and fruit design revealed considerable differences. Compared to south Iberian conspecifics, central European plants were considerably smaller and experienced a highly accelerated generation turnover. In south Iberian populations seed dispersal was carried out almost completely by resident birds which occurred in constant abundances throughout the ripening season. In contrast, central European seeds were dispersed by migrants whose abundances changed considerably during the ripening season. Several bird species were involved in both study areas but rendered different importance for seed dispersal. The fruit ripening pattern was highly asynchronous throughout the ripening season in south Iberia, while central European trees showed a complex ripening sequence which resulted in a significant correlation between fruit abundance and changing disperser availability. Central European fruits were smaller and showed a considerably smaller seed load than south Iberian fruits, thus presumably being more attractive for their small‐sized main dispersers (Sylvia warblers). Chemical analyses revealed significant differences in contents of water, glucose, fructose, proteins, ash. and phenolic compounds. The extensive differentiation of dispersal‐related traits in F. alnus suggests that even weak selective pressures by frugivores may induce evolutionary adjustments of dispersal traits over large time scales. We suggest that the differences we observe today evolved during the species' distribution shifts in the Quaternary.     

Domain–domain motions in proteins from time-modulated pseudocontact shifts

In recent years paramagnetic NMR derived structural constraints have become increasingly popular for the study of biomolecules. Some of these are based on the distance and angular dependences of pseudo contact shifts (PCSs). When modulated by internal motions PCSs also become sensitive reporters on molecular dynamics. We present here an investigation of the domain–domain motion in a two domain protein (PA0128) through time-modulation of PCSs. PA0128 is a protein of unknown function from Pseudomonas aeruginosa (PA) and contains a Zn²⁺ binding site in the N-terminal domain. When substituted with Co²⁺ in the binding site, several resonances from the C-terminal domain showed severe line broadening along the ¹⁵N dimension. Relaxation compensated CPMG experiments revealed that the dramatic increase in the ¹⁵N linewidth came from contributions of chemical exchange. Since several sites with perturbed relaxation are localized to a single β-strand region, and since extracted timescales of motion for the perturbed sites are identical, and since the magnitude of the chemical exchange contributions is consistent with PCSs, the observed rate enhancements are interpreted as the result of concerted domain motion on the timescale of a few milliseconds. Given the predictability of PCS differences and the easy interpretation of the experimental results, we suggest that these effects might be useful in the study of molecular processes occurring on the millisecond to microsecond timescale. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.