Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2.

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1-Gcn2 interaction by binding to Gcn1, thus preventing Gcn2-mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.

Crosslinker length dictates step-growth hydrogel network formation dynamics and allows rapid on-chip photoencapsulation.

Hydrogels formed via free radical-mediated thiol-ene step-growth photopolymerization have been developed for a broad range of tissue engineering and regenerative medicine applications. While the crosslinking mechanism of thiol-ene hydrogels has been well-described, there has been only limited work exploring the physical differences among gels arising from variations in crosslinker properties. Here, we show that the character of linear polyethylene glycol (PEG) dithiols used to crosslink multi-arm polyethylene glycol norbornene (PEGNB) can be used as a facile strategy to tune hydrogel formation kinetics, and therefore the equilibrium hydrogel network architecture. Specifically, we report the dramatic effect of crosslinker length on PEGNB hydrogel formation kinetics and the formed hydrogel properties. It is shown that the hydrogel formation kinetics and formed hydrogel properties can be tuned by solely varying the crosslinker length. It was hypothesized that under identical reaction conditions, a more accessible 3.5 k PEG dithiol crosslinker would improve network ideality relative to a shorter 1.5 k crosslinker. Longer linkers consequently promote significantly more rapid macromer crosslinking and therefore gelation. Accelerated gel formation satisfies an urgent unmet need for rapid polymerization in droplet microfluidics. Using long linkers, we demonstrate the ability to photopolymerize PEGNB microgels under flow on a microfluidic chip, with reliable control over microgel size and shape in a high-throughput manner. To further validate the potential of this platform to produce novel, microstructured cell carrier vehicles, 3T3 fibroblasts were successfully encapsulated and cultured over 14 days with excellent cell viability. This study demonstrates that PEGNB hydrogel dynamics could be readily customized to fulfill a variety of needs in tissue engineering, controlled cell delivery, or drug release applications.

Microbial Mat Communities along an Oxygen Gradient in a Perennially Ice-Covered Antarctic Lake.

Lake Fryxell is a perennially ice-covered lake in the McMurdo Dry Valleys, Antarctica, with a sharp oxycline in a water column that is density stabilized by a gradient in salt concentration. Dissolved oxygen falls from 20 mg liter(-1) to undetectable over one vertical meter from 8.9- to 9.9-m depth. We provide the first description of the benthic mat community that falls within this oxygen gradient on the sloping floor of the lake, using a combination of micro- and macroscopic morphological descriptions, pigment analysis, and 16S rRNA gene bacterial community analysis. Our work focused on three macroscopic mat morphologies that were associated with different parts of the oxygen gradient: (i) "cuspate pinnacles" in the upper hyperoxic zone, which displayed complex topography and were dominated by phycoerythrin-rich cyanobacteria attributable to the genus Leptolyngbya and a diverse but sparse assemblage of pennate diatoms; (ii) a less topographically complex "ridge-pit" mat located immediately above the oxic-anoxic transition containing Leptolyngbya and an increasing abundance of diatoms; and (iii) flat prostrate mats in the upper anoxic zone, dominated by a green cyanobacterium phylogenetically identified as Phormidium pseudopriestleyi and a single diatom, Diadesmis contenta. Zonation of bacteria was by lake depth and by depth into individual mats. Deeper mats had higher abundances of bacteriochlorophylls and anoxygenic phototrophs, including Chlorobi and Chloroflexi. This suggests that microbial communities form assemblages specific to niche-like locations. Mat morphologies, underpinned by cyanobacterial and diatom composition, are the result of local habitat conditions likely defined by irradiance and oxygen and sulfide concentrations.

The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase.

The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.

Dietary protein structure affects endogenous ileal amino acids but not true ileal amino acid digestibility in growing male rats.

BACKGROUND: The amount of endogenous, as opposed to undigested dietary, protein in digesta is a measure of fundamental interest related to gut physiology and function. OBJECTIVE: The objective of this study was to determine whether alimentation with proteins having differing amino acid compositions influenced endogenous ileal amino acids (EIAAs) and true ileal amino acid digestibility (TIAAD) values. METHODS: Male rats (n = 8) were fed a purified diet containing 100 g/kg of 1 of 5 protein hydrolysates, each derived from a different semipurified intact protein source [gelatin, beef muscle (BM), casein, soy protein isolate (SPI), and lactalbumin] devoid of antinutritional factors or fiber. The rats were fed their respective hydrolysate-based diet for 1 d after receiving the same diet but containing the corresponding intact protein source for 7 d. Titanium dioxide was used as an indigestible marker. Ileal digesta were collected after the rats were killed, and EIAAs were determined (precipitate + retentate) after centrifugation and ultrafiltration of the digesta. The TIAAD values of the intact protein sources were determined using EIAA flows based on each protein hydrolysate. RESULTS: Mean EIAA flows differed (P < 0.05) across protein hydrolysates for most amino acids, with the mean ± SEM EIAA flows across amino acids being 262 ± 17, 253 ± 12, 248 ± 18, 226 ± 14, and 191 ± 20 mg/kg dry matter intake for the gelatin, BM, casein, SPI, and lactalbumin hydrolysates, respectively. The only difference (P < 0.05) for the mean EIAA flows across amino acids within each protein hydrolysate was observed between gelatin (262 ± 17 mg/kg) and lactalbumin (191 ± 20 mg/kg) hydrolysates. Except for Trp (P < 0.001) in gelatin and lactalbumin hydrolysates, EIAA flows determined using the casein hydrolysate were not different (P ≥ 0.05) from EIAA flows determined using the other protein hydrolysates. TIAAD values were not generally different (P ≥ 0.05) regardless of the hydrolysate used to determine the EIAA flows. CONCLUSIONS: Protein source affected EIAA flows, although the differences had little effect on TIAAD. Enzyme hydrolyzed casein is a suitable model hydrolysate for determining TIAAD with the enzyme-hydrolyzed protein-ultrafiltration technique.

DPPH radical scavenging activity of a mixture of fatty acids and peptide-containing compounds in a protein hydrolysate of Jatropha curcas seed cake.

Jatropha curcas, a tropical plant, has great potential commercial relevance as its seeds have high oil content. The seeds can be processed into high-quality biofuel producing seed cake as a byproduct. The seed cake, however, has not gotten much attention toward its potential usefulness. This work was aimed to determine the antioxidant activity of different fractions of a protein hydrolysate from J. curcas seed cake and to elucidate the molecular structures of the antioxidants. Seed cake was first processed into crude protein isolate and the protein was hydrolyzed by Neutrase. The hydrolysate obtained from 1 h of Neutrase hydrolysis showed the strongest antioxidant activity against DPPH radical (2,2-diphenyl-1-picrylhydrazyl). After a purification series of protein hydrolysate by liquid chromatography, chemicals acting as DPPH radical inhibitors were found to be a mixture of fatty acids, fatty acid derivatives, and a small amount of peptides characterized by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

NMR and X-ray structures of the putative sterol carrier protein 2 from Thermus thermophilus HB8 show conformational changes.

Sterol carrier protein 2 (SCP-2), also known as nonspecific lipid transfer protein, is a ubiquitous intracellular ~13 kDa protein found in mammals, insects, plants, archaea, and bacteria. Vertebrate SCP-2 has been implicated in a wide range of lipid-related functions in vitro, although its actual physiological role is still unknown. Tunnels in the protein serve as fatty acid binding vehicles. Here we report the first putative SCP-2 structure from a bacterium: specifically, the NMR and X-ray structures of the TTHA0401 protein (also designated as TT1886) from the extremely thermophilic bacterium Thermus thermophilus. The NMR structure and the two chain structures (chain A and chain B) of the asymmetric crystallographic unit (space group (P2(1)2(1)2(1))) revealed an internal cavity. However, this cavity is open to the outside, forming a tunnel, in only one of those structures (chain A, X-ray). The location of this tunnel is different from the one found in other SCP-2 proteins, and inaccessible cavities have not been seen before in SCP structures. We present evidence that at physiological concentrations, TTHA0401 likely exists as a monomer in equilibrium between open and closed conformations. This equilibrium is influenced by temperature-dependent dynamics, and is likely to be very different at the high temperatures preferred by this hyperthermophilic bacterium. Alternatively, another protein binding to TTHA0401 may induce a conformational change, which would constitute an intriguing metabolic regulation method in bacteria.

The NMR solution structures of the five constituent cold-shock domains (CSD) of the human UNR (upstream of N-ras) protein.

Upon cold shock, the amounts of most proteins dramatically decrease from normal levels, but those of cold shock proteins (CSPs) and proteins containing cold-shock domains (CSDs) greatly increase. Although their biological function is still not completely clear, cold-shock proteins might control translation via RNA chaperoning. Many cold-shock proteins contain the motifs (Y/F)GFI and (V/F)(V/F)H, which are known as ribonucleoprotein (RNP)-1 and RNP-2 motifs implicated in RNA/DNA binding. We determined the solution NMR structures of all five constituent CSDs of the human UNR (upstream of N-ras) protein. The spatial arrangements of the sidechains in the RNP-1 and RNP-2 motifs are mostly conserved; however, the conformations of the following residues in the first CSD are different: F43 and H45 (the first phenylalanine residue and the histidine residue in the putative binding site RNP-2) and Y30 (the first residue in the putative binding site RNP-1). F43 and H45 affect each other, and H45 is further influenced by C46. The altered binding site of the first CSD, and its putatively enhanced intrinsic stability, may provide an explanation for the observation that the first CSD has 20-fold higher RNA-binding activity than the fifth CSD. It also lends support to the hypothesis that the UNR protein arose by repeated duplication of a protein that originally contained just one CSD, and that the proto-UNR protein acquired cysteine C46 by mutation during evolution.

NMR solution structures of actin depolymerizing factor homology domains.

Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin-depolymerizing factor homology (ADF-H) domains. ADF-H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best-characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF-H domain of human HIP-55-drebrin-like protein, the first published structure of a drebrin-like domain (mammalian), and the first published structure of GMF beta (mouse). We also determined the structures of mouse GMF gamma, the mouse coactosin-like domain and the C-terminal ADF-H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F-actin) and globular actin (G-actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F-actin binding sites of mouse GMF beta and GMF gamma contain two additional beta-strands not seen in other ADF-H structures. The G-actin binding site of the ADF-H domain of human HIP-55-drebrin-like protein is absent and distorted in mouse GMF beta and GMF gamma.