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Not your typical GPCR Among the large family of G protein–coupled receptors (GPCRs) are many orphans, so called because their signaling reactions remain poorly understood. Among these is GPR158 which is highly expressed in the nervous system and implicated in processes from cognition to memory to mood. Patil et al . determined a high-resolution structure of GPR158 alone and bound to a regulator of G protein signaling (RGS) complex. GPR158 has an unusual dimerization mode with an extensive interaction interface that locks it in a conformation that likely prevents G protein activation. RGS binds to the homodimer at a site that substantially overlaps the surface that binds G proteins, again preventing canonical G protein signaling. Binding of a ligand to the extracellular domain may regulate signaling through the RGS complex. —VV

ATP-BindingCassette (ABC) Transporters employ homologous ATPase domains to drive transmembrane transport of diverse substrates ranging from small molecules to large polymers. Bacterial ABC importers require an extramembranoussubstratebindingprotein (SBP) to deliver the transport substrate to the extracellular side of the transporter complex. Previous studies suggest significant differences in the transport mechanisms of type Ivs. type II bacterial ABC importers, which contain unrelated transmembrane domains. We herein use ensemble fluorescence resonance energy transfer (FRET) experiments to characterize the kinetics of SBP interaction in theE. coliBtuCD-F complex, a canonical type II ABC importer that transports vitamin B12. We demonstrate that, in the absence of B12, BtuF (the SBP) forms a ‘locked’ (kinetically hyper-stable) complex with nanodisc-reconstituted BtuCD that can only be dissociated by ATP hydrolysis, which represents a futile reaction cycle. Notably, no type I importer has been observed to form an equivalent locked complex. We also show that either ATP or vitamin B12binding substantially slows formation of the locked BtuCD-F complex, which will limit the occurrence of futile hydrolysis under physiological conditions.Mutagenesis experiments demonstrate that efficient locking requires concerted interaction of BtuCD with residues on both sides of the B12binding pocket in BtuF. Combined with the kinetic inhibition of locking by ATP binding, these observations imply that the transition state for the locking reaction involves a global alteration in the conformation of BtuCD that extends from its BtuF binding site in the periplasm to its ATP-binding sites on the opposite side of the membrane in the cytoplasm. These observations suggest that locking, which seals the extracellular B12entry site of the transporter, may help push B12through the transporter and directly contribute to the transport mechanism in type II ABC importers.

ATP-BindingCassette (ABC) Transporters use ATP binding and hydrolysis to power transmembrane transport of chemically diverse substrates. Current knowledge of their mechanism comes primarily from static structures of stable intermediates along the transport cycle. Recently, single-molecule fluorescence resonance energy transfer (smFRET) measurements have generated insight into the functional dynamics of transmembrane transporters, but studies to date lack direct information on the physical movement of the transport substrate. Here, we report development of an smFRET system that exploits fluorescence quenching by vitamin B12to track its location in real time during ATP-driven transport by nanodisc-reconstitutedE. coliBtuCD-F, an extensively studied type II ABC importer. Our data demonstrate that transmembrane translocation of B12is driven by two sequential high-energy conformational changes that are inaccessible to standard structural methods because they are inherently transient. The first moves B12from the periplasm into the transmembrane domain of the transporter; notably, this reaction is driven by hydrolysis of a single ATP molecule, in contrast to the mechanism established for several other ABC Transporter families in which ATP-binding drives the mechanochemical power-stroke prior to hydrolysis. The second mediates B12release on the opposite side of the transporter, and it is driven by formation of a hyper-stable complex between BtuCD and BtuF. Hydrolysis of a second single ATP molecule is then required to dissociate BtuCD from the BtuF substrate-binding protein to enable it to bind B12and initiate another round of transport. Our experiments have visualized substrate translocation in real-time at a single-molecule level and provided unprecedented information on the mechanism and dynamics of a paradigmatic transmembrane transport process.

Cystic fibrosis (CF) is caused by mutations in a chloride channel called the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR). We used cryo-EM global conformational ensemble reconstruction to characterize the mechanism by which the breakthrough drug VX445 (Elexacaftor) simultaneously corrects both protein-folding and channel-gating defects caused by CF mutations. VX445 drives hCFTR molecules harboring the gating-defective G551D mutation towards the open-channel conformation by binding to a site in the first transmembrane domain. This binding interaction reverses the usual pathway of allosteric structural communication by which ATP binding activates channel conductance, which is blocked by the G551D mutation. Our ensemble reconstructions include a 3.4 Å non-native structure demonstrating that detachment of the first nucleotide-binding domain of hCFTR is directly coupled to local unfolding of the VX445 binding site. Reversal of this unfolding transition likely contributes to its corrector activity by cooperatively stabilizing NBD1 and the transmembrane domains of hCFTR during biogenesis.SummaryCryo-EM global conformational ensemble reconstruction has been used to characterize the mechanism-of-action of a breakthrough pharmaceutical that corrects fatal protein-folding and channel-gating defects in the human cystic fibrosis transmembrane conductance regulator (CFTR).

The evolutionary benefit accounting for widespread conservation of oligomeric structures in proteins lacking evidence of intersubunit cooperativity remains unclear. Here, crystal and cryo-EM structures, and enzymological data, demonstrate that a conserved tetramer interface maintains the active-site structure in one such class of proteins, the short-chain dehydrogenase/reductase (SDR) superfamily. Phylogenetic comparisons support a significantly longer polypeptide being required to maintain an equivalent active-site structure in the context of a single subunit. Oligomerization therefore enhances evolutionary fitness by reducing the metabolic cost of enzyme biosynthesis. The large surface area of the structure-stabilizing oligomeric interface yields a synergistic gain in fitness by increasing tolerance to activity-enhancing yet destabilizing mutations. We demonstrate that two paralogous SDR superfamily enzymes with different specificities can form mixed heterotetramers that combine their individual enzymological properties. This suggests that oligomerization can also diversify the functions generated by a given metabolic investment, enhancing the fitness advantage provided by this architectural strategy.

Pullulanases are well-known debranching enzymes hydrolyzing α−1,6-glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis-involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the “evolutionary coupling saturation mutagenesis” (ECSM) approach: residue pair co-variances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.

Members of an important group of industrial enzymes, Rhizopus lipases, exhibit valuable hydrolytic features that underlie their biological functions. Particularly important is their N-terminal polypeptide segment (NTPS), which is required for secretion and proper folding but is removed in the process of enzyme maturation. A second common feature of this class of lipases is the α-helical "lid", which regulates the accessibility of the substrate to the enzyme active site. Some Rhizopus lipases also exhibit "interfacial activation" by micelle and/or aggregate surfaces. While it has long been recognized that the NTPS is critical for function, its dynamic features have frustrated efforts to characterize its structure by X-ray crystallography. Here, we combine nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the structure and dynamics of Rhizopus chinensis lipase (RCL) with its 27-residue NTPS prosequence (r27RCL). Both r27RCL and the truncated mature form of RCL (mRCL) exhibit biphasic interfacial activation kinetics with p-nitrophenyl butyrate (pNPB). r27RCL exhibits a substrate binding affinity significantly lower than that of mRCL due to stabilization of the closed lid conformation by the NTPS. In contrast to previous predictions, the NTPS does not enhance lipase activity by increasing surface hydrophobicity but rather inhibits activity by forming conserved interactions with both the closed lid and the core protein structure. Single-site mutations and kinetic studies were used to confirm that the NTPS serves as internal competitive inhibitor and to develop a model of the associated process of interfacial activation. These structure-function studies provide the basis for engineering RCL lipases with enhanced catalytic activities.

YhcB, an uncharacterized protein conserved across gamma-proteobacteria, is composed predominantly of a single Domain of Unknown Function (DUF 1043) with an N-terminal transmembrane α-helix. Here, we show that E. coli YhcB is a conditionally essential protein that interacts with the proteins of the cell divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). We found 7 interactions of YhcB that are conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we identified several point mutations that abolished interactions of YhcB with FtsI and RodZ. The yhcB knock-out strain does not grow at 45°C and is hypersensitive to cell-wall acting antibiotics even in stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septa development. The 2.8 Å crystal structure for the cytosolic domain from Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure that combines parallel and anti-parallel coiled-coil intersubunit interactions. This structure is likely to organize interprotein oligomeric interactions on the inner surface of the cytoplasmic membrane, possibly involved in regulation of cell division and/or envelope biogenesis/integrity in proteobacteria. In summary, YhcB is a conserved and conditionally essential protein that is predicted to play a role in cell division and consequently or in addition affects envelope biogenesis.ImportanceOnly 0.8 % of the protein annotations in the UniProt are based on experimental evidence and thus, functional characterization of unknown proteins remains a rate-limiting step in molecular biology. Herein, the functional properties of YhcB (DUF1043) were investigated using an integrated approach combining X-ray crystallography with genetics and molecular biology. YhcB is a conserved protein that appears to be needed for the transition from exponential to stationary growth and is involved in cell division and/or envelope biogenesis/integrity. This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

Research on the E. coli EttA protein demonstrated that this paradigmatic ABC-F protein controls the first step in polypeptide elongation on the ribosome dependent on cellular energy status. Subsequently, biochemical and structural studies have established that ABC-F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but absent in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. This paper reviews the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC-F proteins, and it discusses the implications of these studies for the molecular function of eukaryotic ABC-F proteins, including the three human representatives of this protein family.

Metalloproteins comprise over one-third of proteins, with approximately half of all enzymes requiring metal to function. Accurate identification of these metal atoms and their environment is a prerequisite to understanding biological mechanism. Using ion beam analysis through particle induced X-ray emission (PIXE), we have quantitatively identified the metal atoms in 30 previously structurally characterized proteins using minimal sample volume and a high-throughput approach. Over half of these metals had been misidentified in the deposited structural models. Some of the PIXE detected metals not seen in the models were explainable as artifacts from promiscuous crystallization reagents. For others, using the correct metal improved the structural models. For multinuclear sites, anomalous diffraction signals enabled the positioning of the correct metals to reveal previously obscured biological information. PIXE is insensitive to the chemical environment, but coupled with experimental diffraction data deposited alongside the structural model it enables validation and potential remediation of metalloprotein models, improving structural and, more importantly, mechanistic knowledge.

The narrow substrate scope of naturally occurring alcohol dehydrogenases (ADHs) greatly limits the enzymatic synthesis of important chiral alcohols. On the basis of X-ray crystal structures and kinetic profiling of a substrate library, we engineered variants of the stereospecific alcohol dehydrogenase from Candida parapsilopsis. This resulted in a set of four mutant enzymes which enable the asymmetric reduction of a broad range of prochiral ketones, including valuable pharmaceuticals and fine chemicals. The engineering strategy of this study paves the way for creating additional ADHs tailored for production of complex chiral alcohols.

Plant-derived cellulose nanofibrils (CNFs) have shown reinforcing effects in polymer nanocomposites. However, freeze-dried CNFs are foam-like material, namely aerogel, that are challenging to disperse in a polymer matrix. In this work, a liquid infusion process was developed for a CNF/epoxy nanocomposite cross-linked foam structure with alterable properties without damaging the foam structure. Microstructures of CNF/epoxy composite foams with different formulations were evaluated using a scanning electron microscope. Surface morphology showed that the CNF cross-linked fibers were well attached by epoxy resin. All absolute and specific mechanical properties [by normalizing the measured parameters against the measured density (ρ)] were investigated. Water resistance and thermal stability of CNF/epoxy composite foams were investigated by water absorption test and thermogravimetric analysis. The concentration of epoxy solution in both tetrahydrofuran (THF) solvents and ethyl acetate (EA) solvents was shown to improve compressive properties and water resistance. The samples fabricated with higher epoxy concentration had higher compressive properties, better water resistance, and better thermal stability. The CNF/epoxy composite foams exhibited compressive modulus and compressive strength up to 175 and 10 MPa, respectively. The water diffusion coefficient of CNF/epoxy composite foams was reduced with an increase in epoxy loading. Further, the CNF/epoxy nanocomposite foams fabricated by the epoxy/THF solution had a more uniform structure and better strength performance than foams fabricated by the epoxy/EA solution, due to the increased solubility of the epoxy in THF compared to epoxy in EA. The glass transition temperature (Tg) was determined by differential scanning calorimetry. The Tg of the nanocomposites was influenced by the CNF/epoxy composition. Therefore, the properties of CNF/epoxy nanocomposite foams can be optimized via changing the solvent and concentration of epoxy resin in solvent.

Wet-formed particleboard panels were made using cellulose nanofibrils (CNF) as the bonding material. The effects of panel density, CNF addition ratio, particle size, and pressing method on the nail and face screw withdrawal strength, water absorption (WA), and thickness swelling (TS) were investigated. The nail and face screw withdrawal strength increased with an increased panel density and CNF addition ratio. Mixed-size particles were favorable for better face screw withdrawal strength. The WA decreased while TS increased with increased panel density. The WA decreased with increased CNF addition ratio. The effect of CNF addition ratio on the TS was influenced by an interaction effect of the particle size, density, and pressing method. Smaller wood particles and the constant thickness (CT) pressing method were better for both WA and TS performance. All of the high- and medium-density panels failed to satisfy the standard requirements for face screw withdrawal strength. For low-density panels, those manufactured with mixed-sized particles all satisfied the standard requirements; those manufactured with large particles required at least 15% CNF to meet the standard, and those manufactured with small particles required at least 20% CNF to achieve the standard requirements. None of the panels met the standard TS requirement (< 8%).

Carbon metabolism of Crabtree-negative yeast Pichia pastoris was profiled using 13C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, 13C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree-negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree-negative Pichia stipitis and Crabtree-positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2H, 13C, 15N-labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR-based structural biology. Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2).

Thermomechanical pulp (TMP) produced using atmospheric refining was ground to isolate lignocellulose nanofibrils (LCNF). Forty minutes and a specific energy of about 1300 kWh/t were necessary to reach the end-point of the process defined by the presence of 95% fines in the slurry. 100% fines were reached after 90 min and 5800 kWh/t. The similarity in structure between pilot-scale produced reference cellulose nanofibrils (CNF) and LCNF with an average fibril diameter of 12 nm confirmed the negligible effect of residual lignin on the defibrillation process. The thermal stability of LCNF was found to be 30 °C lower compared to that of CNF due to the lignin influence, but it was in the range of processing temperatures used for wood composite materials. The effect of using LCNF as adhesive replacement in fiberboard has been assessed. A maximum modulus of rupture of 12.1 MPa comparable to that of commercial fiberboards and high modulus of elasticity, was obtained at 20% LCNF content for panels produced at 180 °C. The binding ability of LCNF and its effect on the mechanical properties of fiberboards were more perceptible when a less refined TMP with lower fine content and longer fibers was used in the process.

This paper presents the experimental behavior of low-energy impact and quasi-static compression test of shifted-tri-axial structural wood-fiber-based composite panels made from laminated paper. The experimental results were analyzed based on design parameters and configurations of panels for the further design and optimization. The results showed that the face stiffness and strength was a significant factor to improve both impact performance and compressive performance. The panels made with additional carbon fiber fabric composite faces had higher energy absorption compared with the same panels made without it. The core configuration also affected the impact behavior of the panels, the foam filled core integrated with the shifted-tri-axial rib structure improved the impact load and absorbed more energy than the same panels without the foam. Further, the structure and size of the element in the core influenced the impact performance and energy absorption. The location for both compression and impact at the triangular lattice element center of the ribs had higher absorbed energy than the location at the hexagonal lattice element center of the ribs. A 3D contour surface map of maximum energy absorption was made based on the experimental data, the contour shows localized energy absorption based on the impact location on the core, the small triangular lattice element of the core had highest maximum energy absorption of panels. For both the quasi-static compression tests and the low-velocity impact tests, the panels with the same core configuration had similar compressive load–displacement trends during the early contact phase. However, the peak load was higher in compression than the peak load for the low-velocity impact for panels with the same configuration.

This paper presents an analysis of 3-dimensional engineered structural panels (3DESP) made from wood-fiber-based laminated paper composites. Since the existing models for calculating the mechanical behavior of core configurations within sandwich panels are very complex, a new simplified orthogonal model (SOM) using an equivalent element has been developed. This model considers both linear and nonlinear geometrical effects when used to analyze the mechanical properties of 3DESP by transforming repeated elements from a tri-axial ribbed core for bending. Two different conditions were studied in comparison with finite element method (FEM) and I-beam equation. The results showed the SOM was consistent with FEM and the experimental result and were more accurate than the I-beam equation. The SOM considering nonlinear geometric deformation needed more computational effort and was found to match well with a FEM model and had slightly better accuracy compared with the linear SOM. Compared with FEM, the parameters in the linear SOM were easier to modify for predicting point-by-point bending performance. However, while the FEM can provide advanced characteristics of the 3DESP such as strain distribution, the linear SOM provided acceptable deformation accuracy and is proposed for preliminary design with multiple parameters. FEM should be applied for advanced analyses.

An important group of industrial enzymes, Rhizopus lipases exhibit valuable hydrolytic features that underlie their biological functions. Particularly important is their N-terminal polypeptide segment (NTPS), which is required for secretion and proper folding, but is removed in the process of enzyme maturation. A second common feature of this class of lipases is the α-helical “lid”, which regulates accessibility of substrate to the enzyme active site. Some Rhizopus lipases also exhibit “interfacial activation” by micelle and/or aggregate surfaces. While it has long been recognized that the NTPS is critical for function, its dynamic features have frustrated efforts to characterize its structure by X-ray crystallography. Here, we combine nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the structure and dynamics of R. chinensis lipase (RCL) with its 27-residue NTPS prosequence (r27RCL). Both r27RCL and the truncated mature form of RCL (mRCL) exhibit bi-phasic interfacial activation kinetics with p-nitrophenyl butyrate (pNPB). r27RCL exhibits a significantly lower substrate binding affinity than mRCL due to stabilization of the closed lid conformation by the NTPS. In contrast to previous predictions, the NTPS does not enhance lipase activity by increasing surface hydrophobicity, but rather inhibits activity by forming conserved interactions with both the closed lid and the core protein structure. Single site-mutations and kinetic studies were used to confirm that the NTPS serves as internal competitive inhibitor, and to develop a model of the associated process of interfacial activation. These structure-function studies provide the basis for engineering RCL lipases with enhanced catalytic activities.

Recombinant proteins are essential for biotechnology. Here we review some of the key points for improving the production of heterologous proteins, and what can be the future of the field.

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival.

This project demonstrates newly invented, biobased construction materials developed by applying low-carbon, biomass waste sources through the Authors' engineered fiber processes and technology. If manufactured and applied large-scale the project inventions can divert large volumes of cellulose waste into high-performance, low embodied energy, environmental construction alternatives that lessen society’s dependence on greenhouse gas emitting products associated with global warming. Biobased materials produced in this work include: 1. High strength-to-weight-ratio "bioboards" as a dense, thin, multi-purpose construction hardboard from 100% waste sources and without resins, binders, or adhesives. Bioboards are used in flat and multidimensional form to create the project inventions. 2. Modular, three-dimensional-core, structural insulated wall, floor, and roof "BioSIPs" panels developed using bioboards as main structural components. BioSIPs received patenting during the project. This publication describes research and computational methodologies, testing of project materials, and their full-scale application as the exterior and interior materials and construction system for a solar-powered case-study building. The authors are currently advancing project inventions for commercialization.

This paper presents static and fatigue bending behavior for a wood-based structural panel having a slot and tab (S/T) construction technique. Comparisons were made with similarly fabricated panels without the S/T construction technique. Experimental results showed that both types of panels had similar bending properties in the static tests. However, the panels with S/T construction had better fatigue results. The failure modes were different for the two fabrication techniques. The panels without S/T debonded at the core:face interface. Whereas, the panels with S/T had cracks that propagated within the rib of the core after debonding damage at the core:face interface. The fatigue deflection-life relationship indicated that the S/T construction improved the connection between the faces and core. The S/T construction decreased the deflection growth rate that delayed panel failure. The fatigue stress-life relationship or degradation was better for the panels with S/T construction than the panels without the S/T construction.

This paper presents a simplified analytical model and balanced design approach for modeling light-weight wood-based structural panels in bending. Because many design parameters are required to input for the model of finite element analysis (FEA) during the preliminary design process and optimization, the equivalent method was developed to analyze the mechanical performance of panels based on experimental results. The bending deflection, normal strain and shear strain of the panels with various configurations were investigated using four point bending test. The results from the analytical model matched well with the experimental data, especially, the prediction for maximum deflection of the panels under failure load. The normal strain and shear strain calculated by the model also agreed with the experimental data. The failure criterion was determined by the failure modes using a 3-dimensional diagram with apparent normal and shear strain. For demonstration, panels 1 and 2 with a fixed core were modeled using the balanced design approach for optimal face thickness. The results showed that both the 3-dimensional diagram and analytical model provided similar thickness results, which were verified by the FEA for wood-based structural panels.

The static and fatigue bending behavior of wood-fiber-based tri-axial engineered sandwich composite panels (ESCP) has been investigated by four-point bending tests. Fatigue panels and weakened panels (wESCP) with an initial interface defect were manufactured for the fatigue tests. Stress σ vs. number of cycles curves (S-N) were recorded under the different stress levels. The primary failure mode in the fatigue tests was observed in the shear zone (epoxy debonding), which was different from face failure in the pure bending zone for the static bending test. For residual bending (RB) test, epoxy debonding failure occurred between the pure bending zone and shear zone. Macro cracks along the core/face interface developed as the number of cycles increased during the fatigue life. The crack propagation or damage for the panels submitted to fatigue test can be described as a three-stage damage process of first non-linear portion, followed by linear damage accumulation, and lastly non-linear accelerated damage. Bending stiffness degradation at the higher load level had faster degradation during fatigue life. The dissipated energy of the panels was small due to the high stiffness of the materials.

In addition to processing a troubling agricultural by-product and reducing demands on our landfills, prepared agro-waste composites are suitable for a variety of practical applications. However, enhancing value-added options for these agricultural by-products can necessitate ability to assess their mechanical integrity. This paper accordingly describes the preparation of a cellulosic-manure composite and demonstrates ability to determine stresses in a perforated structure of the material from measured displacement data. Processing digital image correlation (DIC) recorded displacement information with an Airy stress function gives reliable results full-field as well as at the edge of geometric discontinuities without having to differentiate the recorded data. Required constitutive properties are evaluated in situ and results are substantiated independently.

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.

Wet-formed particleboard bonded with cellulose nanofibrils (CNF) was prepared in this work. The effects of density, CNF addition ratio, pressing method, and particle size on the bending strength were evaluated. The results showed that density had the most important effect on the modulus of elasticity (MOE), while the CNF addition ratio had the most important effect on the modulus of rupture (MOR). For panels with low density ( 800 kg/m3), the pressing method’s effect was more important, compared to panels with low and medium densities. With increased density and CNF addition ratio, panels were able to meet low-density and some medium-density standard MOE and MOR requirements.

This paper presents experimental results of both quasi-static compression and low-velocity impact behavior for tri-axial bio-composite structural panels using a spherical load head. Panels were made having different core and face configurations. The results showed that panels made having either carbon fiber fabric composite faces or a foam-filled core had significantly improved impact and compressive performance over panels without either. Different localized impact responses were observed based on the location of the compression or impact relative to the tri-axial structural core; the core with a smaller structural element had better impact performance. Furthermore, during the early contact phase for both quasi-static compression and low-velocity impact tests, the panels with the same configuration had similar load-displacement responses. The experimental results show basic compression data could be used for the future design and optimization of tri-axial bio-composite structural panels for potential impact applications.

Synonymous codon choices at the beginning of genes optimize 5′ RNA structures for enhanced translation initiation, but less is known about mechanisms that drive codon optimization downstream within the gene. To understand what determines codon choices across a gene, we generated 12,726 in situ codon mutants in the Escherichia coli essential gene infA and measured their fitness by combining multiplex automated genome engineering mutagenesis with amplicon deep sequencing (MAGE-seq). Correlating predicted 5′ RNA structure with fitness revealed that codons even far from the start of the gene are deleterious if they disrupt the native 5′ RNA conformation. These long-range structural interactions generate context-dependent rules that constrain codon choices beyond intrinsic codon preferences. Genome-wide RNA folding predictions confirm that natural codon choices far from the start codon are optimized in part to prevent disruption of native structures near the 5′ UTR. Our results shed light on natural codon distributions and should improve engineering of gene expression for synthetic biology applications.

The TenA protein family occurs in prokaryotes, plants and fungi; it has two subfamilies, one (TenA_C) having an active-site cysteine, the other (TenA_E) not. TenA_C proteins participate in thiamin salvage by hydrolysing the thiamin breakdown product amino-HMP (4-amino-5-aminomethyl-2-methylpyrimidine) to HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine); the function of TenA_E proteins is unknown. Comparative analysis of prokaryote and plant genomes predicted that (i) TenA_E has a salvage role similar to, but not identical with, that of TenA_C and (ii) that TenA_E and TenA_C also have non-salvage roles since they occur in organisms that cannot make thiamin. Recombinant Arabidopsis and maize TenA_E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, hydrolysed the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a line with a null mutation in the HMP biosynthesis gene ThiC prevented its rescue by amino-HMP. Ablating At3g16990 in the wild-type increased sensitivity to paraquat-induced oxidative stress; HMP overcame this increased sensitivity. Furthermore, the expression of TenA_E and ThiC genes in Arabidopsis and maize was inversely correlated. These results indicate that TenA_E proteins mediate amidohydrolase and aminohydrolase steps in the salvage of thiamin breakdown products. As such products can be toxic, TenA_E proteins may also pre-empt toxicity.

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.

Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, Energy-dependent Translational Throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA exit (E) site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA binding (P) site. Using single-molecule fluorescence resonance energy transfer (smFRET), we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, where the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.

ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein energy-dependent translational throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistently with this inference, EttA-deleted cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a new mechanism sensitive to cellular energy status.

Stress and strain concentrations and in-plane and out-of-plane stress constraint factors associated with a circular hole in thick, loaded orthotropic composite plates are determined by three-dimensional finite element method. The plate has essentially infinite in-plane geometry but finite thickness. Results for Sitka spruce wood are emphasized, although some for carbon-epoxy composites are included. While some results are similar to those for isotropy, there are significant consequences due to material orthotropy. Maximum stress and strain concentration factors occur at midplane for thin plates but closer to the external traction-free surfaces for thick plates. These factors decrease as the plate surface is approached and reach lower values unrepresentative of the maximum values. Differences between the midplane and/or maximum and surface stress or strain concentration factors in Sitka spruce, range from 8% if the wood grain is parallel to the vertically applied load to 15% when the grain is perpe...

This paper presents analysis of a 3-dimensional engineered structural panel (3DESP) having a tri-axial core structure made from phenolic impregnated laminated-paper composites with and without high-strength composite carbon-fiber fabric laminated to the outside of both faces. Both I-beam equations and finite element method were used to analyze four-point bending of the panels. Comparisons were made with experimental panels. In this study, four experimental panels were fabricated and analyzed to determine the influence of the carbon-fiber on bending performance. The materials properties for finite element analyses (FEA) and I-beam equations were obtained from either the manufacturer or in-house material tensile tests. The results of the FEA and I-beam equations were used to compare with the experimental 3DESP four-point bending tests. The maximum load, face stresses, shear stresses, and apparent modulus of elasticity were determined. For the I-beam equations, failure was based on maximum stress values. For FEA, the Tsai-Wu strength failure criterion was used to determine structural materials failure. The I-beam equations underestimated the performance of the experimental panels. The FEA-estimated load values were generally higher than the experimental panels exhibiting slightly higher panel properties and load capacity. The addition of carbon-fiber fabric to the face of the panels influenced the failure mechanism from face buckling to panel shear at the face–rib interface. FEA provided the best comparison with the experimental bending results for 3DESP.

While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.

NanoRNase (Nrn) specifically degrades nucleoside 3′,5′-bisphosphate and the very short RNA, nanoRNA, during the final step of mRNA degradation. The crystal structure of Nrn in complex with a reaction product GMP was determined. The overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. Two Mn2+ ions are coordinated by conserved residues in the DHH motif of the N-terminal domain. GMP binds near the DHHA1 motif region in the C-terminal domain. Our structure enables us to predict the substrate-bound form of Nrn as well as other DHH/DHHA1 phosphoesterase family proteins.

The dynamic viscoelasticity of full-size wood composite panels (WCPs) under the free-free vibrational state were determined by a vibration testing method. Vibration detection tests were performed on 194 pieces of three types of full-size WCPs (particleboard, medium density fiberboard, and plywood (PW)). The dynamic viscoelasticity from smaller specimens cut from the panels was measured using a cantilever beam vibration test apparatus, and the two data sets were compared. A strong linear relationship was discovered between the dynamic viscoelasticity values measured by the vibration detection test and the cantilever beam vibration test. The storage modulus values of the panels were far higher than their loss modulus values, and PW panels had the smallest value of loss modulus. For the panels tested, density had a good linear impact on storage modulus. In comparison with density, logarithmic decrement had a greater linear impact on loss modulus. This study demonstrated that the vibration test method is a valid approach for determining the dynamic viscoelasticity of full-size WCPs.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.

The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, but corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 Å resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.