Crystal structure of the invertebrate bifunctional purine biosynthesis enzyme PAICS at 2.8 Å resolution

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5‐aminoimidazole ribonucleotide carboxylase and the 4‐(N‐succinylcarboxamide)‐5‐aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8‐Å resolution crystal structure of the 350‐kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single‐wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species. Proteins 2013; 81:1473–1478. © 2013 Wiley Periodicals, Inc.     

Interacting effects of temperature and density on individual growth performance in a wild population of brown trout

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Harvest selection on multiple traits in the wild revealed by aquatic animal telemetry

Harvesting can have profound impacts on the ecology and evolution of marine populations. However, little is known about the strength and direction of fisheries‐induced selection acting on multiple traits in the wild. Here, we used acoustic telemetry to directly monitor individual behavior and fate in an intensively harvested species, the European lobster (Homarus gammarus, n = 100), in southern Norway. Overall, 24% of the tracked lobsters survived the two‐month harvest season within the study area. Our results indicated that local survival was not random with respect to phenotype. We found no clear support for fisheries‐induced selection acting directly on body size. However, lobsters with large crusher claws relative to their body size, typical of socially dominant individuals, appeared at higher risk of being captured in the conventional trap fishery. We also detected a fine‐scale spatial gradient in survival. After accounting for this gradient, individuals displaying larger home ranges were more likely to survive the harvest season. Finally, we found significant repeatabilities for lobster behavior on a monthly timescale, indicating that individual behavioral attributes tended to persist and may reflect personality. Our study therefore provides empirical support for the need to consider an evolutionary enlightened approach to fisheries management that considers the influence of harvest on multiple traits of target species.     

Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity

Inorganic pyrophosphate (PP(i)) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P(i) in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP(i) are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP(i) on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP(i). This inhibition could be reversed by Tnap, but not if PP(i) was bound to mineral. PP(i) also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP(i) was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP(i), hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP(i) inhibits Tnap-mediated P(i) release from beta-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP(i) prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.     

Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies

The present work shows how UV 'light-induced molecular immobilisation' (LIMI) of biomolecules onto thiol reactive surfaces can be used to make biosensors, without the need for traditional microdispensing technologies. Using 'LIMI,' arrays of biomolecules can be created with a high degree of reproducibility. This technology can be used to circumvent the need for often expensive nano/microdispensing technologies. The ultimate size of the immobilised spots is defined by the focal area of the UV beam, which for a diffraction-limited beam can be less than 1 microm in diameter. LIMI has the added benefit that the immobilised molecules will be spatially oriented and covalently bound to the surface. The activity of the sensor molecules is retained. Antibody sensor arrays made using LIMI demonstrated successful antigen binding. In addition, the pattern of immobilised molecules on the surface is not restricted to conventional array formats. The ultimate consequence of the LIMI is that it is possible to write complex protein patterns using bitmaps at high resolution onto substrates. Thus, LIMI of biomolecules provides a new technological platform for biomolecular immobilisation and the potential for replacing present microdispensing arraying technologies.     

Assessment of chemical-crosslink-assisted protein structure modeling in CASP13

With the advance of experimental procedures obtaining chemical crosslinking information is becoming a fast and routine practice. Information on crosslinks can greatly enhance the accuracy of protein structure modeling. Here, we review the current state of the art in modeling protein structures with the assistance of experimentally determined chemical crosslinks within the framework of the 13th meeting of Critical Assessment of Structure Prediction approaches. This largest-to-date blind assessment reveals benefits of using data assistance in difficult to model protein structure prediction cases. However, in a broader context, it also suggests that with the unprecedented advance in accuracy to predict contacts in recent years, experimental crosslinks will be useful only if their specificity and accuracy further improved and they are better integrated into computational workflows.     

Crystal structure of an archaeal class I aldolase and the evolution of (betaalpha)8 barrel proteins

Fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in the glycolytic pathway. FBPAs from archaeal organisms have recently been identified and characterized as a divergent family of proteins. Here, we report the first crystal structure of an archaeal FBPA at 1.9-A resolution. The structure of this 280-kDa protein complex was determined using single wavelength anomalous dispersion followed by 10-fold non-crystallographic symmetry averaging and refined to an R-factor of 14.9% (Rfree 17.9%). The protein forms a dimer of pentamers, consisting of subunits adopting the ubiquitous (betaalpha)8 barrel fold. Additionally, a crystal structure of the archaeal FBPA covalently bound to dihydroxyacetone phosphate was solved at 2.1-A resolution. Comparison of the active site residues with those of classical FBPAs, which share no significant sequence identity but display the same overall fold, reveals a common ancestry between these two families of FBPAs. Structural comparisons, furthermore, establish an evolutionary link to the triosephosphate isomerases, a superfamily hitherto considered independent from the superfamily of aldolases.