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A terbium-based metal-organic framework, namely {[Tb

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were C-terminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.

Proteins of halophilic organisms, which accumulate molar concentrations of KCl in their cytoplasm, have a much higher content in acidic amino acids than proteins of mesophilic organisms. It has been proposed that this excess is necessary to maintain proteins hydrated in an environment with low water activity, either via direct interactions between water and the carboxylate groups of acidic amino acids or via cooperative interactions between acidic amino acids and hydrated cations. Our simulation study of five halophilic proteins and five mesophilic counterparts does not support either possibility. The simulations use the AMBER ff14SB force field with newly optimized Lennard-Jones parameters for the interactions between carboxylate groups and potassium ions. We find that proteins with a larger fraction of acidic amino acids indeed have higher hydration levels, as measured by the concentration of water in their hydration shell and the number of water/protein hydrogen bonds. However, the hydration level of each protein is identical at low (bKCl = 0.15 mol/kg) and high (bKCl = 2 mol/kg) KCl concentrations; excess acidic amino acids are clearly not necessary to maintain proteins hydrated at high salt concentration. It has also been proposed that cooperative interactions between acidic amino acids in halophilic proteins and hydrated cations stabilize the folded protein structure and would lead to slower dynamics of the solvation shell. We find that the translational dynamics of the solvation shell is barely distinguishable between halophilic and mesophilic proteins; if such a cooperative effect exists, it does not have that entropic signature.

Background Candidatus Nanohaloarchaeota, an archaeal phylum within the DPANN superphylum, is characterized by limited metabolic capabilities and limited phylogenetic diversity and until recently has been considered to exclusively inhabit hypersaline environments due to an obligate association with Halobacteria. Aside from hypersaline environments, Ca. Nanohaloarchaeota can also have been discovered from deep-subsurface marine sediments. Results Three metagenome-assembled genomes (MAGs) representing a new order within the Ca. Nanohaloarchaeota were reconstructed from a stratified salt crust and proposed to represent a novel order, Nucleotidisoterales. Genomic features reveal them to be anaerobes capable of catabolizing nucleotides by coupling nucleotide salvage pathways with lower glycolysis to yield free energy. Comparative genomics demonstrated that these and other Ca. Nanohaloarchaeota inhabiting saline habitats use a “salt-in” strategy to maintain osmotic pressure based on the high proportion of acidic amino acids. In contrast, previously described Ca. Nanohaloarchaeota MAGs from geothermal environments were enriched with basic amino acids to counter heat stress. Evolutionary history reconstruction revealed that functional differentiation of energy conservation strategies drove diversification within Ca. Nanohaloarchaeota, further leading to shifts in the catabolic strategy from nucleotide degradation within deeper lineages to polysaccharide degradation within shallow lineages. Conclusions This study provides deeper insight into the ecological functions and evolution of the expanded phylum Ca. Nanohaloarchaeota and further advances our understanding on the functional and genetic associations between potential symbionts and hosts.

PURPOSE OF REVIEW: Compared to the current standard of implanting bone anabolics for fracture repair, bone fracture targeted anabolics would be more effective, less invasive, less toxic and would allow for control over what phase of fracture healing is being affected. We therefore sought to identify the optimal bone targeting molecule to allow for systemic administration of therapeutics to bone fractures. RECENT FINDINGS: We found that many bone targeting molecules exist, but most have been developed for the treatment of bone cancers, osteomyelitis, or osteoporosis. There are a few examples of bone targeting ligands that have been developed for bone fractures that are selective for the bone fracture over the body and skeleton. SUMMARY: Acidic oligopeptides have the ideal half-life, toxicity profile, and selectivity for a bone fracture targeting ligand and are the most developed and promising of these bone fracture targeting ligands. However, many other promising ligands have been developed that could be used for bone fractures.

PURPOSE OF REVIEW: The skeletal system provides an important role to support body structure and protect organs. The complexity of its architecture and components makes it challenging to deliver the right amount of the drug into bone regions, particularly avascular cartilage lesions. In this review, we describe the recent advance of bone-targeting methods using bisphosphonates, polymeric oligopeptides, and nanoparticles on osteoporosis and rare skeletal diseases. RECENT FINDINGS: Hydroxyapatite (HA), a calcium phosphate with the formula Ca(10)(PO(4))(6)(OH)(2), is a primary matrix of bone mineral that includes a high concentration of positively charged calcium ion and is found only in bone. This unique feature makes HA a general targeting moiety to the entire skeletal system. We have applied bone-targeting strategy using acidic amino acid oligopeptides into lysosomal enzymes, demonstrating the effects of bone-targeting enzyme replacement therapy and gene therapy on bone and cartilage lesions in inherited skeletal disorders. Virus or no-virus gene therapy using techniques of engineered capsid or nanomedicine has been studied preclinically for skeletal diseases. SUMMARY: Efficient drug delivery into bone lesions remains an unmet challenge in clinical practice. Bone targeting therapies based on gene transfer can be potential as new candidates for skeletal diseases.

In modern agriculture, frequent application of herbicides may induce the evolution of resistance in plants, but the mechanisms underlying herbicide resistance remain largely unexplored. Here, we report the characterization of rtp1 (resistant to paraquat 1), an Arabidopsis mutant showing strong resistance to the widely used herbicides paraquat and diquat. The rtp1 mutant is semi-dominant and carries a point mutation in the gene encoding the multidrug and toxic compound extrusion family protein DTX6, leading to the change of glycine to glutamic acid at residue 311 (G311E). The wild-type DTX6 with glycine 311 conferred weak paraquat and diquat resistance when overexpressed, while mutation of glycine 311 to a negatively charged amino acid (G311E or G311D) markedly increased the paraquat and diquat resistance of plants, whereas mutation to a positively charged amino acid (G311R or G311K) compromised the resistance, suggesting that the charge property of residue 311 of DTX6 is critical for the paraquat and diquat resistance of Arabidopsis plants. DTX6 is localized in the endomembrane trafficking system and may undergo the endosomal sorting to localize to the vacuole and plasma membrane. Treatment with the V-ATPase inhibitor ConA reduced the paraquat resistance of the rtp1 mutant. Paraquat release and uptake assays demonstrated that DTX6 is involved in both exocytosis and vacuolar sequestration of paraquat. DTX6 and DTX5 show functional redundancy as the dtx5 dtx6 double mutant but not the dtx6 single mutant plants were more sensitive to paraquat and diquat than the wild-type plants. Collectively, our work reveals a potential mechanism for the evolution of herbicide resistance in weeds and provides a promising gene for the manipulation of plant herbicide resistance.

Antimicrobial resistance (AMR) is emerging as a global threat to public health. One of the strategies employed to combat AMR is the use of adjuvants which act to enhance or reinstate antimicrobial activity by inhibiting resistance mechanisms. However, these adjuvants are themselves not immune to selecting resistant phenotypes. Thus, there is a need to utilise mechanisms which are either less likely to or unable to trigger resistance. One commonly employed mechanism of resistance by microorganisms is to prevent antimicrobial uptake or efflux the antibiotic which manages to permeate its membrane. Here we propose amino acids as antimicrobial adjuvants that may be utilizing alternate mechanisms to fight AMR. We used a modified ethidium bromide (EtBr) efflux assay to determine its efflux in the presence of ciprofloxacin within Staphylococcus aureus (NCTC 8325) and Pseudomonas aeruginosa (PAO1). In this study, aspartic acid and glutamic acid were found to inhibit growth of both bacterial species. Moreover, a reduced production of toxic pigments, pyocyanin and pyoverdine by P. aeruginosa was also observed. As evident from similar findings with tetracycline, these adjuvants, may be a way forward towards tackling antimicrobial resistance.

Recently, substantial levels of acidic D-amino acids, such as D-aspartate and D-glutamate, have been identified in many organisms, from bacteria to mammals, suggesting that acidic D-amino acids have multiple physiological significances. Although acidic D-amino acids found in animals primarily originate from foodstuffs and/or bacteria, the D-aspartate-synthesizing enzyme aspartate racemase is identified in various animals. In eukaryotic organisms, acidic D-amino acids are primarily degraded by the flavoenzyme D-aspartate oxidase (DDO). DDO is found in multiple eukaryotic organisms and may play important roles in acidic D-amino acid utilization, elimination, and intracellular level regulation. Moreover, owing to its perfect enantioselectivity and stereoselectivity, DDO may be a valuable tool in several biotechnological applications, including the identification and quantification of acidic D-amino acids. In this mini-review, previous DDO reports are summarized and the potential bioengineering and biotechnological applications of DDO are discussed. Key Points ・Occurrence and distribution ofd-aspartate oxidase. ・Fundamental properties of d -aspartate oxidase of various eukaryotic organisms. ・Biotechnological applications and potential engineering ofd-aspartate oxidase.

The effects of trisodium citrate (TSC) and disodium tartrate (DST) based food preservatives on the hydration behaviors of the amino acids L-aspartic acid (ASP) and L-glutamic acid (GLU) have been studied using thermodynamic, transport, calorimetric and spectroscopic studies. The volumetric, acoustic and viscosity data suggest that hydrophilic–ionic/hydrophilic interactions are predominant in these systems. The calculated parameters are worthwhile for exploring the solutes as structure-breakers, and the solutes undergo pairwise interactions with the co-solutes. The sweetness of both amino acids decreases in the presence of the preservatives. The hydration number and solvation data suggest that these solutes are more hydrated in water. The dominance of dehydration effects in relation to TSC is observed from the positive enthalpy changes in calorimetry studies and the negative chemical shifts in 1H NMR studies.

The objective of this paper is to investigate the potential affecting mechanisms of l-lysine (Lys)/l-arginine (Arg) on myosin solubility. The results showed that both Lys and Arg increased the solubility of myosin at the examined pH values. Additionally, both Lys and Arg decreased the hydrodynamic size of myosin but increased the hydration capacity (HC), the surface aromatic hydrophobicity of myosin, the surface tension of the myosin solution and the absolute transfer free energy (TFE) of the major amino acids that constitute myosin. The results indicate that the properties of Lys or Arg that result in an inhibition of myosin aggregation and an interaction with hydrophobic amino acid residues may play important roles in increasing the myosin solubility. The results are attractive to the meat industry.

The electrostatic (ES) energy of each residue was for the first time quantitatively evaluated in a flavin mononucleotide binding protein (FBP). A residue electrostatic energy (RES) was obtained as the sum of the ES energies between atoms in each residue and all other atoms in the FBP dimer using atomic coordinates obtained by a molecular dynamics (MD) simulation. ES is one of the most important energies among the interaction energies in a protein. It is determined from the RES, the residues which mainly contribute to stabilize the structure of each subunit, and the binding energy between two subunits can be estimated. The RES of all residues in subunit A (Sub A) and subunit B (Sub B) were attractive forces, even though the residues contain net negative or positive charges. This reveals that the ES energies of any of the residues can contribute to stabilize the protein structure. The total binding ES energy over all residues among the subunits was distributed between −0.2 to −1.2 eV (mean = −0.67 eV) from the MD simulation time.

Background Bio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics. Results Under aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture. Conclusions Our results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives. Graphical Abstract

Acetylation of non-histone proteins plays important roles in regulating protein functions but the mechanisms of action are poorly understood. Our recent study uncovered a previously unknown mechanism by which C-terminal domain (CTD) acetylation of p53 serves as a “switch” to determine the interaction between a unique group of acidic domain-containing proteins and p53, as well as revealed that acidic domains may act as a novel class of “readers” for unacetylated p53. However, the properties of acidic domain “readers” are not well elucidated yet. Here, we identified that the charge effect between acidic domain “readers” and the p53 CTD is necessary for their interaction. Both the length and the amino acid composition of a given acidic domain contributed to its ability to recognize the p53 CTD. Finally, we summarized the characteristic features of our identified acidic domains, which would distinguish this kind of “readers” from other types of acidic amino acid-containing domains.

Chirality is ubiquitous in biology, including in biomineralization, where it is found in many hardened structures of invertebrate marine and terrestrial organisms (for example, spiralling gastropod shells). Here we show that chiral, hierarchically organized architectures for calcium carbonate (vaterite) can be controlled simply by adding chiral acidic amino acids (Asp and Glu). Chiral, vaterite toroidal suprastructure having a ‘right-handed' (counterclockwise) spiralling morphology is induced by L-enantiomers of Asp and Glu, whereas ‘left-handed' (clockwise) morphology is induced by D-enantiomers, and sequentially switching between amino-acid enantiomers causes a switch in chirality. Nanoparticle tilting after binding of chiral amino acids is proposed as a chiral growth mechanism, where a ‘mother' subunit nanoparticle spawns a slightly tilted, consequential ‘daughter' nanoparticle, which by amplification over various length scales creates oriented mineral platelets and chiral vaterite suprastructures. These findings suggest a molecular mechanism for how biomineralization-related enantiomers might exert hierarchical control to form extended chiral suprastructures., Chiral structures are formed in numerous processes including biomineralization of calcium carbonate. Here, the authors demonstrate that the chiral, hierarchically-organized architecture of the calcium carbonate mineral, vaterite, can be controlled simply by the addition of chiral acidic amino acids.

Mammalian Na

We investigated if the therapeutic switching of sofalcone (SFC), a gastroprotective agent, to an anticolitic agent is feasible using colon-targeted drug delivery. SFC can activate the anti-inflammatory nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-hemeoxygenase-1 (HO-1) pathway in human colon epithelial cells and murine macrophages. For the efficient treatment of colitis, SFC was coupled with acidic amino acids to yield SFC-aspartic acid (SFC-AA) and SFC-glutamic acid, and their colon targetability and therapeutic effects were assessed as an anticolitic agent in a 2,4-dinitrobenezenesulfonic acid-induced rat colitis model. The SFC derivatives were decoupled up to 72% in the cecal contents but remained stable in the small intestinal contents. Oral gavage of SFC-AA (oral SFC-AA, equivalent to 1.67 mg/kg of SFC) delivered SFC (maximal cecal concentration: 57.36 μM) to the cecum, while no SFC was detected with oral gavage of SFC (oral SFC, 1.67 mg/kg). Moreover, oral SFC-AA (equivalent to 10 mg/kg of SFC) did not afford detectable concentration of SFC in the blood but detected up to 4.64 μM with oral SFC (10 mg/kg), indicating efficient colonic delivery and limited systemic absorption of SFC upon oral SFC-AA. Oral SFC-AA ameliorated colonic damage and inflammation in rat colitis with elevating colonic levels of HO-1 and nuclear Nrf2 protein, and the anticolitic effects of SFC-AA were significantly undermined by an HO-1 inhibitor. At an equivalent dose of SFC, oral SFC-AA but not oral SFC increased colonic HO-1 and nuclear Nrf2 levels, and oral SFC-AA was more effective than oral SFC in treating rat colitis. Moreover, oral SFC-AA was as effective against colitis as oral sulfasalazine being used for the treatment of inflammatory bowel disease. In conclusion, colon-targeted delivery of SFC facilitated the therapeutic switching of the drug to an anticolitic drug via Nrf2 activation.

Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle., Odinstova, Mohammed, Trieber et al. use structure-based mutagenesis to identify key residues in human periplakin and desmoplakin linker modules required for co-localization with vimentin intermediate filaments. They show that vimentin D176 and E187 are required for direct binding with the periplakin linker.

The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sodium in exchange for an extracellular proton. We examined the functional role of acidic amino acids of a yeast specific periplasmic extracellular loop 6 (EL6) and of Glu74 and Arg77 of transmembrane segment 3. Glu74 and Arg77 are conserved in yeast species while Glu74 is conserved throughout various phyla. The mutation E74A caused a minor effect, while mutation R77A had a larger effect on the ability of SpNHE1 to confer salt tolerance. Mutation of both residues to Ala or Glu also eliminated the ability to confer salt tolerance. Arg341 and Arg342 were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp389, Glu390, Glu392, Glu397) of EL6 did not greatly affect SpNHE1 function while deletion of all did. Replacement of EL6 with a segment from the plant Na+/H+ exchanger SOS1 also did not affect function. We suggest that EL6 forms part of a cation coordination sphere, attracting cations for transport but that the region is not highly specific for the location of acidic charges. Overall, we identified a number of polar amino acids important in SpNHE1 function.

Surface charge engineering of nanosized CuS via acidic amino acid modification is proved to be an efficient strategy to realize high peroxidase-mimicking catalytic activity at neutral pH. As a proof-of-concept application, one-pot high-performance colorimetric sensing of glucose by coupling the engineered nanozyme with glucose oxidase is realized.

In this study, a lab-scale multiple draft tubes airlift loop membrane bioreactor (Mt-ALMBR) was used for treating acidic 7-amino cephalosporanic acid (7-ACA) wastewater under different pHs (3.54–6.20) and hydraulic retention time (HRT) (48 h, 36 h, 24 h and 16 h). During about 200 days operation, under HRT of 48 h and pH condition about 6.0, the optimum average COD and BOD5 removal rates were reach to 84.4 ± 2.1% and 94.9 ± 0.8%, and the highest 7-ACA removal rate also observed as 77.6%. Biodegradation, membrane rejection, hydrolysis and sludge adsorption were the four main pathways of 7-ACA removal. With the increase of pH, biodegradation, membrane rejection and hydrolysis had significant positive impacts on 7-ACA removal, while adsorption had a negative impact. Moreover, mathematical models for 7-ACA removal rate and pH were calculated to guide the operation of Mt-ALMBR. Biodegradation was the main pathway to remove 7-ACA when pH was >4.17.

Intracellular pH level plays an important role in physiological and pathological processes. The development of nanoprobes for detecting in vivo pH levels is especially important for early diagnosis of disease. Therefore, we develop a hydrophilic carbon points (CDs) using quercetin and ethylenediamine as precursors to monitor intracellular pH. Under optimized conditions, the prepared CDs not only have uniform particle size and morphology, but also possess strong green fluorescence, photostability, and photoreversibility in water medium. The CDs exhibit pH-sensitive fluorescence effect under acidic and alkaline conditions, which is used to achieve "off-on-off" detection pH (from 3.5 to 13.5). Meanwhile, the pH-dependent mechanism is further investigated and explained, which is the fluorescence quenching caused by the pH-induced aggregation. Based on the pH-sensitive characteristics of CDs, it has been applied to the detection of aspartic acid and glutamic acid. More importantly, when applied to live cells, the pH-probe exhibits low cytotoxicity and high sensitivity, and is successfully used in intracellular pH fluorescence imaging. Consequently, this nanoprobe is expected to be used for real-time monitoring of intracellular pH level.

Adequate protein intake is crucial for the survival and well-being of animals. How animals assess prospective protein sources and ensure dietary amino acid intake plays a critical role in protein homeostasis. By using a quantitative feeding assay, we show that three amino acids, L-glutamate (L-Glu), L-alanine (L-Ala) and L-aspartate (L-Asp), but not their D-enantiomers or the other 17 natural L-amino acids combined, rapidly promote food consumption in the fruit fly Drosophila melanogaster. This feeding-promoting effect of dietary amino acids is independent of mating experience and internal nutritional status. In vivo and ex vivo calcium imagings show that six brain neurons expressing diuretic hormone 44 (DH44) can be rapidly and directly activated by these amino acids, suggesting that these neurons are an amino acid sensor. Genetic inactivation of DH44+ neurons abolishes the increase in food consumption induced by dietary amino acids, whereas genetic activation of these neurons is sufficient to promote feeding, suggesting that DH44+ neurons mediate the effect of dietary amino acids to promote food consumption. Single-cell transcriptome analysis and immunostaining reveal that a putative amino acid transporter, CG13248, is enriched in DH44+ neurons. Knocking down CG13248 expression in DH44+ neurons blocks the increase in food consumption and eliminates calcium responses induced by dietary amino acids. Therefore, these data identify DH44+ neuron as a key sensor to detect amino acids and to enhance food intake via a putative transporter CG13248. These results shed critical light on the regulation of protein homeostasis at organismal levels by the nervous system.

Nanomaterials have triggered tremendous interest to mimick peroxidase but rarely attention has been paid to small molecules. Herein we first found that acidic amino acids including l -glutamic acid (L-Glu) and l -aspartic acid (L-Asp) exhibited an intrinsic peroxidase-like activity, endowing acidic amino acids with the capability of catalysing the oxidation of the peroxidase substrates 3,3′,5,5′-tetramethylbenzidine (TMB) to produce color reaction in the presence of H2O2. Reaction mechanism was further investigated by means of electron spin resonance spectroscopy (ESR), enzyme kinetics assay and quantum theoretical calculations, to verify and provide a good deal of insight into the catalytic process. Based on the above discovery, a colorimetric platform was successfully developed for sensing glucose in the range of 0.10 μM to 10 μM with a detection limit of 40 nM, as well as evaluating the inhibitory effect of antioxidants on reactive oxygen species. This extraordinary finding not only extends the new biological function of acidic amino acids, but also opens new opportunities to deepen the knowledge of the new class of small molecule enzymes.

The current study aimed to investigate the association between dietary amino acid patterns and incidence of hypertension, using principal components factor analyses. This study was conducted within the framework of Tehran Lipid and Glucose Study on 4288 adults, who were free of hypertension at baseline (2008–2011) and were followed for three years (2011–2014). Principal component factor analyses were conducted based on eight amino acid groups and three amino acid patterns were extracted. The first pattern was characterized by branched chain, aromatic, and alcoholic amino acids, and proline. Acidic amino acids and proline were highly loaded in the second pattern and the third was characterized by sulphuric and small amino acids. Adjusted odds ratio of the highest quartile of the first pattern was 1.83 (95%CI: 1.21–2.77, P for trend = 0.002) compared to the lowest one. The first pattern had high positive correlation with dietary intakes of animal protein and dairy, but was negatively correlated with plant protein, fruit, and vegetable. There was no significant association for the second and third patterns. Findings indicate that the dietary amino acid pattern, rich in branched chain, aromatic, and alcoholic amino acids, and proline could increase the risk of hypertension.

In situ fabrication of carbonated hydroxyapatite (CHA) remineralization layer on an enamel slice was completed in a novel, biomimetic two-step method. First, a CaCO 3 layer was synthesized on the surface of demineralized enamel using an acidic amino acid (aspartic acid or glutamate acid) as a soft template. Second, at the same concentration of the acidic amino acid, rod-like carbonated hydroxyapatite was produced with the CaCO 3 layer as a sacrificial template and a reactant. The morphology, crystallinity and other physicochemical properties of the crystals were characterized using field emission scanning electron microscopy (FESEM), Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy-dispersive X-ray analysis (EDAX), respectively. Acidic amino acid could promote the uniform deposition of hydroxyapatite with rod-like crystals via absorption of phosphate and carbonate ions from the reaction solution. Moreover, compared with hydroxyapatite crystals coated on the enamel when synthesized by a one-step method, the CaCO 3 coating that was synthesized in the first step acted as an active bridge layer and sacrificial template. It played a vital role in orienting the artificial coating layer through the template effect. The results show that the rod-like carbonated hydroxyapatite crystals grow into bundles, which are similar in size and appearance to prisms in human enamel, when using the two-step method with either aspartic acid or acidic glutamate (20.00 mmol/L).

CRSBP-1 (mammalian LYVE-1) is a membrane glycoprotein highly expressed in lymphatic endothelial cells (LECs). It has multiple ligands, including hyaluronic acid (HA) and growth factors/cytokines (e.g., PDGF-BB and VEGF-A) containing CRS motifs (clusters of basic amino-acid residues). The ligand binding activities are mediated by Link module and acidic-amino-acid-rich (AAAR) domains, respectively. These CRSBP-1/LYVE-1 ligands have been shown to induce opening of lymphatic intercellular junctions in LEC monolayers and in lymphatic vessels in wild-type mice. We hypothesize that CRSBP-1/LYVE-1 ligands, particularly CRS-containing growth factors/cytokines, are secreted by immune and cancer cells for lymphatic entry during adaptive immune responses and lymphatic metastasis. We have looked into the origin of the Link module and AAAR domain of LYVE-1 in evolution and its association with the development of lymph nodes and efficient adaptive immunity. Lymph nodes represent the only major recent innovation of the adaptive immune systems in evolution particularly to mammals and bird. Here we demonstrate that the development of the LYVE-1 gene with the AAAR domain in evolution is associated with acquisition of lymph nodes and adaptive immunity. LYVE-1 from other species, which have no lymph nodes, lack the AAAR domain and efficient adaptive immunity. Synthetic CRSBP-1 ligands PDGF and VEGF peptides, which contain the CRS motifs of PDGF-BB and VEGF-A, respectively, specifically bind to CRSBP-1 but do not interact with either PDGFβR or VEGFR2. These peptides function as adjuvants by enhancing adaptive immunity of pseudorabies virus (PRV) vaccine in pigs. These results support the notion that LYVE-1 is involved in adaptive immunity in mammals.

ADP-ribosylation, a posttranslational modification catalyzed by a family of enzymes known as poly(ADP- ribose) polymerases (PARPs, 17 in humans), regulates diverse cellular processes. To aid in understanding the functions of ADP-ribosylation in cells, we developed a clickable aminooxy probe, AO-alkyne, which detects ADP-ribosylation of acidic amino acids. AO-alkyne can be used to detect auto-ADP-ribosylation of PARP10 in cells following Cu-catalyzed click conjugation to an azide reporter. This method can be extended to other PARP family members that catalyze ADP-ribosylation on acidic amino acids, providing a convenient and direct readout of PARP activity in cells.

Aryl-keto-containing α-amino acids are of great importance in organic chemistry and biochemistry. They are valuable intermediates for the construction of hydroxyl α-amino acids, nonproteinogenic α-amino acids, as well as other biofunctional components. Friedel-Crafts acylation is an effective method to prepare aryl-keto derivatives. In this review, we summarize the preparation of aryl-keto containing α-amino acids by Friedel-Crafts acylation using acidic α-amino acids as acyl-donors and Lewis acids or Brönsted acids as catalysts.

Halophilic proteins are stable and function at high salt concentration. Understanding how these molecules maintain their fold stable and avoid aggregation under harsh conditions is of great interest for biotechnological applications. This mini-review describes what is known about the molecular determinants of protein halotolerance. Comparisons between the sequences of halophilic/non-halophilic homologous protein pairs indicated that Asp and Glu are significantly more frequent, while Lys, Ile and Leu are less frequent in halophilic proteins. Homologous halophilic and non-halophilic proteins have similar overall structure, secondary structure content, and number of residues involved in the formation of H-bonds. On the other hand, on the halophilic protein surface, a decrease of nonpolar residues and an increase of charged residues are observed. Particularly, halophilic adaptation correlates with an increase of Asp and Glu, compensated by a decrease of basic residues, mainly Lys, on protein surface. A thermodynamic model, that provides a reliable explanation of the salt effect on the conformational stability of globular proteins, is presented.

Conserved acidic and aromatic residues in the four sodium channel voltage-sensor domains make domain-specific functional contributions., Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation–pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in NaV VSDs.

Protein mutations can result in dysfunctional cell signaling pathways; therefore it is of significance to develop a robust platform for the detection of protein mutations. Here, we report that the channel of bacterial virus T7 DNA packaging motor is able to discriminate peptides containing a mixture of acidic (negatively charged) and basic (positively charged) amino acids. Peptides were differentiated based on their current signatures created by their unique charge compositions. In combination with protease digestion, peptides with the locational differences of single amino acid were also identified. The results suggest that the T7 motor channel has the potential for peptide differentiation, mutation verification, and analysis of protein sequence.

β-Sheets are one of the fundamental three-dimensional building blocks for protein structures. Oppositely charged amino acids are frequently observed directly across one another in antiparallel sheet structures, suggesting the importance of cross-strand ion pairing interactions. Despite the apparent electrostatic nature of ion pairing interactions, the charged amino acids Asp, Glu, Arg, Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. Accordingly, the effect of charged amino acid side chain length on cross-strand ion pairing interactions at lateral non-hydrogen bonded positions was investigated in a β-hairpin motif. The negatively charged residues with a carboxylate (Asp, Glu, Aad in increasing length) were incorporated at position 4, and the positively charged residues with an ammonium (Dap, Dab, Orn, Lys in increasing length) were incorporated at position 9. The fraction folded population and folding free energy were derived from the chemical shift deviation data. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Only the Asp/Glu-Dap interactions with shorter side chains and the Aad-Orn/Lys interactions with longer side chains exhibited stabilizing energetics, mostly relying on electrostatics and hydrophobics, respectively. This suggested the need for length matching of the interacting residues to stabilize the β-hairpin motif. A survey of a nonredundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-LysGlu-Lys, also implying the need for length matching of the oppositely charged residues.

Database survey in this study revealed that about one-third of the protein structures deposited in the Protein Data Bank (PDB) contain arginine-arginine (Arg-Arg) pairing with a carbon···carbon (CZ···CZ) interaction distance less than 5 Å. All the Arg-Arg pairings were found to bury in a polar environment composed of acidic residues, water molecules, and strong polarizable or negatively charged moieties from binding site or bound ligand. Most of the Arg-Arg pairings are solvent exposed and 68.3% Arg-Arg pairings are stabilized by acidic residues, forming Arg-Arg-Asp/Glu clusters. Density functional theory (DFT) was then employed to study the effect of environment on the pairing structures. It was revealed that Arg-Arg pairings become thermodynamically stable (about -1 kcal/mol) as the dielectric constant increases to 46.8 (DMSO), in good agreement with the results of the PDB survey. DFT calculations also demonstrated that perpendicular Arg-Arg pairing structures are favorable in low dielectric constant environment, while in high dielectric constant environment parallel structures are favorable. Additionally, the acidic residues can stabilize the Arg-Arg pairing structures to a large degree. Energy decomposition analysis of Arg-Arg pairings and Arg-Arg-Asp/Glu clusters showed that both solvation and electrostatic energies contribute significantly to their stability. The results reported herein should be very helpful for understanding Arg-Arg pairing and its application in drug design.

Summary Heat stress transcription factors (HSFs) compose a large gene family, and different members play differential roles in regulating plant responses to abiotic stress. The objectives of this study were to identify and characterize an A2‐type HSF, FaHsfA2c, in a cool‐season perennial grass tall fescue (Festuca arundinacea Schreb.) for its association with heat tolerance and to determine the underlying physiological functions and regulatory mechanisms of FaHsfA2c imparting plant tolerance to heat stress. FaHsfA2c was localized in nucleus and exhibited a rapid transcriptional increase in leaves and roots during early phase of heat stress. Ectopic expression of FaHsfA2c improved basal and acquired thermotolerance in wild‐type Arabidopsis and also restored heat‐sensitive deficiency of hsfa2 mutant. Overexpression of FaHsfA2c in tall fescue enhanced plant tolerance to heat by triggering transcriptional regulation of heat‐protective gene expression, improving photosynthetic capacity and maintaining plant growth under heat stress. Our results indicated that FaHsfA2c acted as a positive regulator conferring thermotolerance improvement in Arabidopsis and tall fescue, and it could be potentially used as a candidate gene for genetic modification and molecular breeding to develop heat‐tolerant cool‐season grass species.

Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg 2+ ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N -gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.

We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.

Full or partial burial of ionizable groups in the hydrophobic interior of proteins underlies the large modulation in group properties (modified pK value, high nucleophilicity, enhanced capability of interaction with chemical moieties of the substrate, etc.) linked to biological function. Indeed, the few internal ionizable residues found in proteins are known to play important functional roles in catalysis and, in general, in energy transduction processes. However, ionizable-group burial is expected to be seriously disruptive and, it is important to note, most functional sites contain not just one, but several ionizable residues. Hence, the adaptations involved in the development of function in proteins (through in vitro engineering or during the course of natural evolution) are not fully understood. Here, we explore experimentally how proteins respond to the accumulation of hydrophobic-to-ionizable residue substitutions. For this purpose, we have constructed a combinatorial library targeting a hydrophobic cluster in a consensus-engineered, stabilized form of a small model protein. Contrary to naive expectation, half of the variants randomly selected from the library are soluble, folded, and active, despite including up to four mutations. Furthermore, for these variants, the dependence of stability with the number of mutations is not synergistic and catastrophic, but smooth and approximately linear. Clearly, stabilized protein scaffolds may be robust enough to withstand many disruptive hydrophobic-to-ionizable residue mutations, even when they are introduced in the same region of the structure. These results should be relevant for protein engineering and may have implications for the understanding of the early evolution of enzymes. Proteins 2012; © 2011 Wiley Periodicals, Inc.

Hsp40-like co-chaperones are ubiquitous enzymes that stimulate the protein refolding activity of Hsp70 family chaperones. They are widespread in prokaryotic and eukaryotic systems. In bacteria, the best characterized co-chaperone is the Escherichia coli DnaJ protein. Many γ-proteobacteria encode a functional homologue of DnaJ, known as CbpA, which is expressed in response to starvation and environmental stress. The activity of CbpA is regulated by the “modulator” protein CbpM. Here, we have used a combination of genetics and biochemistry to identify the co-chaperone contact determinant of CbpM. We show that the nature of the interaction is conserved in enterobacteria.

Many ion channels have been shown to be regulated by the membrane signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here, we demonstrate that the binding of PIP(2) to SpIH, a sea urchin hyperpolarization-activated cyclic nucleotide-gated ion channel (HCN), has a dual effect: potentiation and inhibition. The potentiation is observed as a shift in the voltage dependence of activation to more depolarized voltages. The inhibition is observed as a reduction in the currents elicited by the partial agonist cGMP. These two effects were separable and arose from PIP(2) binding to two different regions. Deletion of the C-terminal region of SpIH removed PIP(2)-induced inhibition but not the PIP(2)-induced shift in voltage dependence. Mutating key positively charged amino acids in the C-terminal region adjacent to the membrane selectively disrupted PIP(2)-induced inhibition, suggesting a direct interaction between PIP(2) in the membrane and amino acids in the C-terminal region that stabilizes the closed state relative to the open state in HCN channels.

Analysis of trace amino acids (AA) in physiological fluids has received more attention, because the analysis of these compounds could provide fundamental and important information for medical, biological, and clinical researches. More accurate method for the determination of those compounds is highly desirable and valuable. In the present study, we developed a selective and sensitive method for trace AA determination in biological samples using 2-[2-(7H-dibenzo [a,g]carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC) as labeling reagent by HPLC-FLD-MS/MS. Response surface methodology (RSM) was first employed to optimize the derivatization reaction between DBCEC and AA. Compared with traditional single-factor design, RSM was capable of lessening laborious, time and reagents consumption. The complete derivatization can be achieved within 6.3 min at room temperature. In conjunction with a gradient elution, a baseline resolution of 20 AA containing acidic, neutral, and basic AA was achieved on a reversed-phase Hypersil BDS C(18) column. This method showed excellent reproducibility and correlation coefficient, and offered the exciting detection limits of 0.19-1.17 fmol/μL. The developed method was successfully applied to determinate AA in human serum. The sensitive and prognostic index of serum AA for liver diseases has also been discussed.

The P2Y1 receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y1 receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or di-hydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins.

Deletion of Phe508 (DeltaF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. The mutation severely reduces the stability and folding of the protein by disrupting interactions between NBD1 and the second transmembrane domain (TMD2). We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of DeltaF508-CFTR with V510D more efficiently than V510E. Promotion of DeltaF508-CFTR maturation did not require NBD2 as introduction of V510D into a DeltaNBD2/DeltaF508-CFTR mutant restored maturation to levels similar to that of full-length protein. The V510D mutation increased the half-life of mature DeltaF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. It was also observed that introduction of the V510R/R1070D mutations into DeltaF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. We propose that the V510D mutation in NBD1 promotes maturation and stabilizes DeltaF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2.

In order to investigate the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety as a carboxylic acid bioisoster at ionotropic glutamate receptors (iGluRs), a series of acidic alpha-aminocarboxylic acids in which the distal carboxy group was replaced by the 4-hydroxy-1,2,5-oxadiazol-3-yl group was synthesized. Ionization constants were determined. All target compounds, except the Asp analogue 12, were resolved using chiral HPLC. Whereas 12 showed good affinity exclusively at NMDA receptors, the Glu analogue, (+)-10, was an unselective, though potent AMPA receptor preferring agonist (EC(50) = 10 microM at iGluR2) showing only low stereoselectivity. The two higher Glu homologues, (+)-15 and (+)-18, turned out to be weak agonists at iGluR2 as well as weak antagonists at NR1/NR2A, whereas the corresponding (-)-isomers were selective NR1/NR2A antagonists with somewhat higher potency. The results proved the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety to be a useful bioisoster at all three classes of iGluRs, capable of being integrated into agonists as well as antagonists.