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Over the last few decades, efforts to transition the global production of fuels and chemicals toward renewable carbon feedstocks have accelerated. A large portion of these efforts have focused on valorization of one of the most abundant renewable carbon sources, lignocellulose. Pretreatment of lignocellulose is the first critical step in this process. In this study, novel ionic liquid (IL) systems consisting of multiple ions known to be effective at biomass pretreatment were tested on woody and grassy biomass. Molecular simulations and experimental results established the synergistic advantages of combining specific individual components in these systems. For pine (woody) biomass, pretreatment with the combination of imidazolium, cholinium, acetate, and lysinate ions achieved 80% glucose and 70% xylose yields at high biomass loading. For sorghum biomass, an IL system comprising cholinium, lysinate, and palmitate ions not only enabled a 98% glucose yield but was also found to be biocompatible in a one-pot configuration, producing the biofuel precursor bisabolene using an engineered strain of the yeast Rhodosporidium toruloides.

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity-and, by extension, the myriad effects of ubiquitination on substrate proteins-arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate's C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

NsaS is one of four intramembrane histidine kinases (HKs) in Staphylococcus aureus that mediate the pathogen's response to membrane active antimicrobials and human innate immunity. We describe the first integrative structural study of NsaS using a combination of solution state NMR spectroscopy, chemical-cross-linking, molecular modeling and dynamics. Three key structural features emerge: First, NsaS has a short N-terminal amphiphilic helix that anchors its transmembrane (TM) bundle into the inner leaflet of the membrane such that it might sense neighboring proteins or membrane deformations. Second, the transmembrane domain of NsaS is a 4-helix bundle with significant dynamics and structural deformations at the membrane interface. Third, the intracellular linker connecting the TM domain to the cytoplasmic catalytic domains of NsaS is a marginally stable helical dimer, with one state likely to be a coiled-coil. Data from chemical shifts, heteronuclear NOE, H/D exchange measurements and molecular modeling suggest that this linker might adopt different conformations during antibiotic induced signaling.

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl-CoA thioesterase (FlK) from Streptomyces cattleya thus provides a unique opportunity to study an enzyme-ligand pair that has been evolutionarily optimized for a surprisingly high 106 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl-CoA and acetyl-CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (KD). Although the magnitude of discrimination is similar to that found in designed synthetic ligand-protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity.

Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants.

Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ(54) activators. Two NtrC4-binding sites were identified upstream (-145 and -85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ(54) regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis.

We compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ(54) transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ(54)-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-like homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly.

Hexavalent chromium [Cr(VI)] is a worldwide water contaminant that is currently without cost-effective and efficient remediation strategies. This is in part due to a lack of ligands that can bind it amid an excess of innocuous ions in aqueous solution. We present herein the design and application of a peptoid-based library of ligand candidates for toxic metal ions. A selective screening process was used to identify members of the library that can bind to Cr(VI) species at neutral pH and in the presence of a large excess of spectator ions. There were 11 sequences identified, and their affinities were compared using titrations monitored with UV-vis spectroscopy. To identify the interactions involved in coordination and specificity, we evaluated the effects of sequence substitutions and backbone variation in the highest affinity structure. Additional characterization of the complex formed between this sequence and Cr(VI) was performed using NMR spectroscopy. To evaluate the ability of the developed sequences to remediate contaminated solutions, the structures were synthesized on a solid-phase resin and incubated with environmental water samples that contained simulated levels of chromium contamination. The synthetic structures demonstrated the ability to reduce the amount of toxic chromium to levels within the range of the EPA contamination guidelines. In addition to providing some of the first selective ligands for Cr(VI), these studies highlight the promise of peptoid sequences as easily prepared components of environmental remediation materials.

BackgroundMild brain hypothermia (32°-34°C) after human neonatal asphyxia improves neurodevelopmental outcomes. Astrocytes but not neurons have pyruvate carboxylase and an acetate uptake transporter. C nuclear magnetic resonance spectroscopy of rodent brain extracts after administering [1-C]glucose and [1,2-C]acetate can distinguish metabolic differences between glia and neurons, and tricarboxylic acid cycle entry via pyruvate dehydrogenase and pyruvate carboxylase.MethodsNeonatal rat cerebrocortical slices receiving a C-acetate/glucose mixture underwent a 45-min asphyxia simulation via oxygen-glucose-deprivation followed by 6 h of recovery. Protocols in three groups of N=3 experiments were identical except for temperature management. The three temperature groups were: normothermia (37°C), hypothermia (32°C for 3.75 h beginning at oxygen--glucose deprivation start), and delayed hypothermia (32°C for 3.75 h, beginning 15 min after oxygen-glucose deprivation start). Multivariate analysis of nuclear magnetic resonance metabolite quantifications included principal component analyses and the L1-penalized regularized regression algorithm known as the least absolute shrinkage and selection operator.ResultsThe most significant metabolite difference (P

Agarobiose (AB; d-galactose-β-1,4-AHG), produced by one-step acid hydrolysis of agarose of red seaweed, is considered a promising cosmetic ingredient due to its skin-moisturizing activity. In this study, the use of AB as a cosmetic ingredient was found to be hampered due to its instability at high temperature and alkaline pH. Therefore, to increase the chemical stability of AB, we devised a novel process to synthesize ethyl-agarobioside (ethyl-AB) from the acid-catalyzed alcoholysis of agarose. This process mimics the generation of ethyl α-glucoside and glyceryl α-glucoside by alcoholysis in the presence of ethanol and glycerol during the traditional Japanese sake-brewing process. Ethyl-AB also showed in vitro skin-moisturizing activity similar to that of AB, but showed higher thermal and pH stability than AB. This is the first report of ethyl-AB, a novel compound produced from red seaweed, as a functional cosmetic ingredient with high chemical stability. [ABSTRACT FROM AUTHOR]

The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of [sup.18]O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter. doi: 10.1128/JB.00201-10

The study is conducted to determine two-component signal transduction as a predominant information processing mechanism in prokaryotes and is present in single-cell eukaryotes and higher plants. The result reveals that solution structures of the apo and activated DctD receiver domain derived from small angle scattered data and corroborate the previously proposed model for how receiver domains regulates the downstream domains.

The hydrolytic catalysis of the mutant subtilisin, Bacillus protease N', with a low-barrier hydrogen bond between the aspartate and histidine residues, is about 8-fold reduced than that of wild-type enzyme. Data indicate that the hydrogen bond is not required for enhanced activity of subtilisin.

Amide hydrogen exchange in the acid state, quenching and indirect nuclear magnetic resonance detection of amide protons were performed consecutively to investigate the acid state structure of the Escherichia coli ribonuclease RNase H*. The mixed alpha + beta protein, which has all its free cysteines substituted with alanine, exhibits a molten globule state in the acid state. Consequently, it was found that the alpha-helical subdomain of the native structure influences its stability and structure under acidic conditions.

Titration calorimetric and circular dichroism spectroscopic methods enable the determination of enthalpic and entropic contributions of two distamycin ligands in the formation of a side-by-side complex formed by the interaction of the minor groove ligand distamycin with the central A3T2 binding site of a DNA duplex. Higher van der Waals attractions account for the more favorable entropy associated with the binding of the second distamycin molecule. Ionic strength significantly influences the binding of distamycin ligands.

The 15N-1H correlation spectra of 15N-labeled unphosphorylated and phosphorylated forms of III (sup Glc) protein from Escherichia coli were compared and assigned NH, 15N and 13Calpha through nuclear magnetic resonance examination. The use of different correlation spectroscopy enabled the assignments of 1H and 13Cbeta signals to the backbone and side-chains of the protein without employing the nuclear Overhauser effect data. Four segments near the amino acids in the active sites exhibited chemical shifts that indicated the restriction of structural changes during phosphorylation in these four sites.