Structures of single‐layer β‐sheet proteins evolved from β‐hairpin repeats

Free‐standing single‐layer β‐sheets are extremely rare in naturally occurring proteins, even though β‐sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single‐layer, anti‐parallel β‐sheet proteins, comprised of three or four twisted β‐hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β‐sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent‐exposed tyrosine residues, was identified on the concave surface of the β‐sheet. These new modular single‐layer β‐sheet proteins may serve as a new model system for studying folding and design of β‐rich proteins.     

Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED

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Plasma mammalian leptin analogue predicts reproductive phenology,but not reproductive output in a capital‐income breeding seaduck

To invest in energetically demanding life history stages, individuals require a substantial amount of resources. Physiological traits, particularly those related to energetics, can be useful for examining variation in life history decisions and trade‐offs because they result from individual responses to environmental variation. Leptin is a protein hormone found in mammals that is proportional to the amount of endogenous fat stores within an individual. Recently, researchers have confirmed that a mammalian leptin analogue (MLA), based on the mammalian sequence of leptin, is present with associated receptors and proteins in avian species, with an inhibitory effect on foraging and body mass gain at high circulating levels. While MLA has been both quantified and manipulated in avian species, little is currently known regarding whether plasma MLA in wild‐living species and individuals is associated with key reproductive decisions. We quantified plasma MLA in wild, Arctic‐nesting female common eiders (Somateria mollissima) at arrival on the breeding grounds and followed them to determine subsequent breeding propensity, and reproductive phenology, investment, and success. Common eiders are capital‐income breeding birds that require the accumulation of substantial fat stores to initiate laying and successfully complete incubation. We found that females with lower plasma MLA initiated breeding earlier and in a shorter period of time. However, we found no links between plasma MLA levels and breeding propensity, clutch size, or reproductive success. Although little is still known about plasma MLA, based on these results and its role in influencing foraging behaviors and condition gain, plasma MLA appears to be closely linked to reproductive timing and is therefore likely to underlie trade‐offs surrounding life history decisions.     

Metal complexation of chitosan and its glutaraldehyde cross-linked derivative

The physicochemical characterization of metal complexed with chitosan (CS) and its glutaraldehyde cross-linked derivative (CSGA) was investigated. Seven metal ions from chromium through zinc of the first row of the transition metals were selected for complexation. Structural features pertinent to where and how metals bind into both polymers are our main interest. Studies using solid-state NMR spectroscopy and XRPD (X-ray powder diffraction) supported by ESR spectroscopy, ICP-OES (inductively couple plasma-optical emission spectroscopy) and far-FTIR spectroscopy for metal interaction with nitrogen sites at C-2 of the metal-polymer complexes were performed. Theoretical calculations of the metal-polymer ratio, the approximate charges on nitrogen for both amine and imino-linker, and the proton affinity between an alcohol group from the polymer and an amino/imino group are reported. A helical coiled chitosan model and a 2C1L (two-chitosans with one linker) model are proposed here. The metal uptake mechanism for both polymers is concluded to be absorption within the polymers, rather than adsorption on the polymer surface.     

RAC3 down-regulation sensitizes human chronic myeloid leukemia cells to TRAIL-induced apoptosis

The nuclear receptor coactivator RAC3 plays important roles in many biological processes and tumorigenesis. We found that RAC3 is over-expressed in human chronic myeloid leukemia cells K562, which are normally resistant to TRAIL-induced apoptosis. RAC3 down-regulation by siRNA rendered these cells sensitive to TRAIL-induced cell death. In addition to the up-regulation of TRAIL receptors, the process involves Bid, caspases and PARP activation, loss of mitochondrial membrane potential, and release of AIF, cytochrome c and Smac/DIABLO to the cytoplasm. We conclude that RAC3 is required for TRAIL resistance and that this anti-apoptotic function is independent of its role in hormone receptor signaling.     

Molecular population genetics of the male and female mitochondrial DNA molecules of the California sea mussel, Mytilus californianus

The presence of two gender-associated mitochondrial genomes in marine mussels provides a unique opportunity to investigate the dynamics of mtDNA evolution without complications inherent in interspecific comparisons. Here, we assess the relative importance of selection, mutation, and differential constraint in shaping the patterns of polymorphism within and divergence between the male (M) and female (F) mitochondrial genomes of the California sea mussel, Mytilus californianus. Partial sequences were obtained from homologous regions of four genes (nad2, cox1, atp6, and nad5) totaling 2307 bp in length. The M and F mtDNA molecules of M. californianus exhibited extensive levels of nucleotide polymorphism and were more highly diverged than observed in other mytilids (overall Tamura-Nei distances >40%). Consistent with previous studies, the M molecule had significantly higher levels of silent and replacement polymorphism relative to F. Both genomes possessed large numbers of singleton and low-frequency mutations that gave rise to significantly negative Tajima's D values. Mutation-rate scalars estimated for silent and replacement mutations were elevated in the M genome but were not sufficient to account for its higher level of polymorphism. McDonald-Kreitman tests were highly significant at all loci due to excess numbers of fixed replacement mutations between molecules. Strong purifying selection was evident in both genomes in keeping the majority of replacement mutations at low population frequencies but appeared to be slightly relaxed in M. Our results suggest that a reduction in selective constraint acting on the M genome remains the best explanation for its greater levels of polymorphism and faster rate of evolution.     

Inhibition of the dopamine D1 receptor signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.     

3-D ultrastructure of O. tauri: electron cryotomography of an entire eukaryotic cell

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes.