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Acidic CO2 electrolysis, enhanced by the introduction of alkali cations, presents a strategic approach for improving carbon efficiency compared to processes conducted in neutral and alkaline environments. However, a significant challenge arises from the dissolution of both organic acids and alkali cations in a strongly acidic feed stream, resulting in a considerable energy penalty for downstream separation. In this study, we investigate the feasibility of using flow-electrode capacitive deionization (FCDI) technology to separate organic acids and recover alkali cations from a strongly acidic feed stream (pH ~ 1). We show that organic acids, such as formic acid and acetic acid, are retained in molecular form in the separation chamber, achieving a rejection rate of over 90% under all conditions. Alkali cations, such as K+ and Cs+, migrate to the cathode chamber in ionic form, with their removal and recovery significantly influenced by their concentration and the pH of the feed stream, but responding differently to the types and concentrations of organic acids. The energy consumption for the removal and recovery of K+ is 4 to 8 times higher than for Cs+, and the charge efficiency is significantly influenced by the types of organic acid products and alkali cations. We conduct a series of electrochemical measurements and analyze the impedance spectroscopy, identifying that hindered mass transfer governed the electrode process. Our findings underscore the potential of FCDI as an advanced downstream separation technology for acidic electrocatalysis processes. [ABSTRACT FROM AUTHOR]

The increase of CO2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO2 as a feedstock gains an extensive importance to establish CO2-neutral or CO2-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO2, to support sustainability of industrial bioprocesses. [ABSTRACT FROM AUTHOR]

The gas-phase formation of new particles less than 1 nm in size and their subsequent growth significantly alters the availability of cloud condensation nuclei (CCN, >30-50 nm), leading to impacts on cloud reflectance and the global radiative budget. However, this growth cannot be accounted for by condensation of typical species driving the initial nucleation. Here, we present evidence that nucleated iodine oxide clusters provide unique sites for the accelerated growth of organic vapors to overcome the coagulation sink. Heterogeneous reactions form low-volatility organic acids and alkylaminium salts in the particle phase, while further oligomerization of small a-dicarbonyls (e.g., glyoxal) drives the particle growth. This identified heterogeneous mechanism explains the occurrence of particle production events at organic vapor concentrations almost an order of magnitude lower than those required for growth via condensation alone. A notable fraction of iodine associated with these growing particles is recycled back into the gas phase, suggesting an effective transport mechanism for iodine to remote regions, acting as a "catalyst" for nucleation and subsequent new particle production in marine air. [ABSTRACT FROM AUTHOR]

Bacteria have evolved multiple signal transduction systems that permit an adaptation to changing environmental conditions. Chemoreceptor-based signaling cascades are very abundant in bacteria and are among the most complex signaling systems. Currently, our knowledge on the molecular features that determine signal recognition at chemoreceptors is limited. Chemoreceptor McpA of Bacillus velezensis SQR9 has been shown to mediate chemotaxis to a broad range of different ligands. Here we show that its ligand binding domain binds directly 13 chemoattractants. We provide support that organic acids and amino acids bind to the membrane-distal and membrane-proximal module of the dCache domain, respectively, whereas binding of sugars/sugar alcohols occurred at both modules. Structural biology studies combined with site-directed mutagenesis experiments have permitted to identify 10 amino acid residues that play key roles in the recognition of multiple ligands. Residues in membrane-distal and membrane-proximal regions were central for sensing organic acids and amimo acids, respectively, whereas all residues participated in sugars/sugar alcohol sensing. Most characterized chemoreceptors possess a narrow and well-defined ligand spectrum. We propose here a sensing mechanism involving both dCache modules that allows the integration of very diverse signals by a single chemoreceptor. [ABSTRACT FROM AUTHOR]

Two-dimensional van der Waals materials have rich and unique functional properties, but many are susceptible to corrosion under ambient conditions. Here we show that linear alkylamines n-CmH2m+1NH2, with m = 4 through 11, are highly effective in protecting the optoelectronic properties of these materials, such as black phosphorus (BP) and transition-metal dichalcogenides (TMDs: WS2, 1T'-MoTe2, WTe2, WSe2, TaS2, and NbSe2). As a representative example, n-hexylamine (m = 6) can be applied in the form of thin molecular monolayers on BP flakes with less than 2-nm thickness and can prolong BP's lifetime from a few hours to several weeks and even months in ambient environments. Characterizations combined with our theoretical analysis show that the thin monolayers selectively sift out water molecules, forming a drying layer to achieve the passivation of the protected 2D materials. The monolayer coating is also stable in air, H2 annealing, and organic solvents, but can be removed by certain organic acids. [ABSTRACT FROM AUTHOR]

Iron oxyhydroxide minerals, known to be chemically reactive and significant for elemental cycling, are thought to have been abundant in early-Earth seawater, sediments, and hydrothermal systems. In the anoxic Fe2+-rich early oceans, these minerals would have been only partially oxidized and thus redox-active, perhaps able to promote prebiotic chemical reactions. We show that pyruvate, a simple organic molecule that can form in hydrothermal systems, can undergo reductive amination in the presence of mixed-valence iron oxyhydroxides to form the amino acid alanine, as well as the reduced product lactate. Furthermore, geochemical gradients of pH, redox, and temperature in iron oxyhydroxide systems affect product selectivity. The maximum yield of alanine was observed when the iron oxyhydroxide mineral contained 1:1 Fe(II):Fe(III), under alkaline conditions, and at moderately warm temperatures. These represent conditions that may be found, for example, in iron-containing sediments near an alkaline hydrothermal vent system. The partially oxidized state of the precipitate was significant in promoting amino acid formation: Purely ferrous hydroxides did not drive reductive amination but instead promoted pyruvate reduction to lactate, and ferric hydroxides did not result in any reaction. Prebiotic chemistry driven by redoxactive iron hydroxide minerals on the early Earth would therefore be strongly affected by geochemical gradients of Eh, pH, and temperature, and liquid-phase products would be able to diffuse to other conditions within the sediment column to participate in further reactions. [ABSTRACT FROM AUTHOR]

Hypochlorous acid (HOCl) is generated in the immune system to kill microorganisms. In Escherichia coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). Here, we identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, we identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand-binding site in the RD. Furthermore, we proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudopalindromic motifs that are separated by ~100 bp, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms. [ABSTRACT FROM AUTHOR]

Members of the aquaporin (AQP) family have been suggested to transport aluminum (Al) in plants; however, the Al form transported by AQPs and the roles of AQPs in Al tolerance remain elusive. Here we report that NIP1;2, a plasma membrane-localized member of the Arabidopsis nodulin 26-like intrinsic protein (NIP) subfamily of the AQP family, facilitates Al-malate transport from the root cell wall into the root symplasm, with subsequent Al xylem loading and root-to-shoot translocation, which are critical steps in an internal Al tolerance mechanism in Arabidopsis. We found that NIP1;2 transcripts are expressed mainly in the root tips, and that this expression is enhanced by Al but not by other metal stresses. Mutations in NIP1;2 lead to hyperaccumulation of toxic Al3+ in the root cell wall, inhibition of root-to-shoot Al translocation, and a significant reduction in Al tolerance. NIP1;2 facilitates the transport of Al-malate, but not Al3+ ions, in both yeast and Arabidopsis. We demonstrate that the formation of the Al-malate complex in the root tip apoplast is a prerequisite for NIP1;2-mediated Al removal from the root cell wall, and that this requires a functional root malate exudation system mediated by the Al-activated malate transporter, ALMT1. Taken together, these findings reveal a critical linkage between the previously identified Al exclusion mechanism based on root malate release and an internal Al tolerance mechanism identified here through the coordinated function of NIP1;2 and ALMT1, which is required for Al removal from the root cell wall, root-to-shoot Al translocation, and overall Al tolerance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Creatine, a nitrogenous organic acid, replenishes cytoplasmic ATP at the expense of mitochondrial ATP via the phosphocreatine shuttle. Creatine levels are maintained by diet and endogenous synthesis from arginine and glycine. Glycine amidinotransferase (GATM) catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from arginine to glycine to form ornithine and guanidinoacetate. We screened 36,530 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea–mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). Among 27 colitis susceptibility phenotypes identified and mapped, one was strongly correlated with a missense mutation in Gatm in a recessive model of inheritance, and causation was confirmed by CRISPR/Cas9 gene targeting. Supplementation of homozygous Gatm mutants with exogenous creatine ameliorated the colitis phenotype. CRISPR/Cas9- targeted (Gatmc/c) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatmc/c mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatmc/c colonocytes showed increased metabolic stress in response to DSS with higher levels of phospho-AMPK and lower levels of phosphorylation of mammalian target of rapamycin (phospho-mTOR). These findings establish an in vivo requirement for rapid replenishment of cytoplasmic ATP within colonic epithelial cells in the maintenance of the mucosal barrier after injury. [ABSTRACT FROM AUTHOR]

Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomainmisfolds slowdown folding by an order ofmagnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of crossdomain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo. [ABSTRACT FROM AUTHOR]

Anion channelrhodopsins (ACRs) are a class of light-gated channels recently identified in cryptophyte algae that provide unprecedented fast and powerful hyperpolarizing tools for optogenetics. Analysis of photocurrents generated by Guillardia theta ACR 1 (GtACR1) and its mutants in response to laser flashes showed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH and involving different amino acid residues. The first mechanism, characterized by slow opening and fast closing of the channel, is regulated by Glu-68. Neutralization of this residue (the E68Q mutation) specifically suppressed this first mechanism, but did not eliminate it completely at high pH. Our data indicate the involvement of another, yetunidentified pH-sensitive group X. Introducing a positive charge at the Glu-68 site (the E68R mutation) inverted the channel gating so that it was open in the dark and closed in the light, without altering its ion selectivity. The second mechanism, characterized by fast opening and slow closing of the channel, was not substantially affected by the E68Q mutation, but was controlled by Cys-102. The C102A mutation reduced the rate of channel closing by the second mechanism by ~100-fold, whereas it had only a twofold effect on the rate of the first. The results show that anion conductance by ACRs has a fundamentally different structural basis than the relatively well studied conductance by cation channelrhodopsins (CCRs), not attributable to simply a modification of the CCR selectivity filter. [ABSTRACT FROM AUTHOR]

Northern permafrost soils store a vast reservoir of carbon, nearly twice that of the present atmosphere. Current and projected climate warming threatens widespread thaw of these frozen, organic carbon (OC)-rich soils. Upon thaw, mobilized permafrost OC in dissolved and particulate forms can enter streams and rivers, which are important processors of OC and conduits for carbon dioxide (CO2) to the atmosphere. Here, we demonstrate that ancient dissolved organic carbon (DOC) leached from 35,800 y B.P. permafrost soils is rapidly mineralized to CO2. During 200-h experiments in a novel high-temporal-resolution bioreactor, DOC concentration decreased by an average of 53%, fueling a more than sevenfold increase in dissolved inorganic carbon (DIC) concentration. Eighty-seven percent of the DOC loss to microbial uptake was derived from the low-molecular-weight (LMW) organic acids acetate and butyrate. To our knowledge, our study is the first to directly quantify high CO2 production rates from permafrost-derived LMW DOC mineralization. The observed DOC loss rates are among the highest reported for permafrost carbon and demonstrate the potential importance of LMW DOC in driving the rapid metabolism of Pleistocene-age permafrost carbon upon thaw and the outgassing of CO2 to the atmosphere by soils and nearby inland waters. [ABSTRACT FROM AUTHOR]

Microbial corrosion textures in volcanic glass from Cenozoic seafloor basalts and the corresponding titanite replacement microtextures in metamorphosed Paleoarchean pillow lavas have been interpreted as evidence for a deep biosphere dating back in time through the earliest periods of preserved life on earth. This interpretation has been recently challenged for Paleoarchean titanite replacement textures based on textural and geochronological data from pillow lavas in the Hooggenoeg Complex of the Barberton Greenstone Belt in South Africa. We use this controversy to explore the strengths and weaknesses of arguments made in support or rejection of the biogenicity interpretation of bioalteration trace fossils in Cenozoic basalt glasses and their putative equivalents in Paleoarchean greenstones. Our analysis suggests that biogenicity cannot be taken for granted for all titanite-based textures in metamorphosed basalt glass, but a cautious and critical evaluation of evidence suggests that biogenicity remains the most likely interpretation for previously described titanite microtextures in Paleoarchean pillow lavas. [ABSTRACT FROM AUTHOR]

Paired immunoglobulin-like type 2 receptor α (PILRα) and β (PILRβ) belong to the PILR family and are related to innate immune regulation in various species. Despite their high sequence identity, PILRα and PILRβ are shown to have variant sialic acid (SA) binding avidities. To explore the molecular basis of this interaction, we solved the crystal structures of PILRα and PILRβ at resolutions of 1.6 Å and 2.2 Å, respectively. Both molecules adopt a typical siglec fold but use a hydrophobic bond to substitute the siglecspecific disulfide linkage for protein stabilization. We further used HSV-1 glycoprotein B (gB) as a representative molecule to study the PILR-SA interaction. Deploying site-directed mutagenesis, we demonstrated that three residues (Y2, R95, and W108) presented on the surface of PILRα form the SA binding site equivalent to those in siglecs but are arranged in a unique linear mode. PILRβ differs from PILRα in one of these three residues (L108), explaining its inability to engage gB. Mutation of L108 to tryptophan in PILRβ restored the gB-binding capacity. We further solved the structure of this PILRβ mutant complexed with SA, which reveals the atomic details mediating PILR/SA recognition. In comparison with the free PILR structures, amino acid Y2 oriented variantly in the complex structure, thereby disrupting the linear arrangement of PILR residues Y2, R95, and W108. In conclusion, our study provides significant implications for the PILR-SA interaction and paves the way for understanding PILR-related ligand binding. [ABSTRACT FROM AUTHOR]

The increase of CO 2 emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO 2 as a feedstock gains an extensive importance to establish CO 2 -neutral or CO 2 -negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii ( Pichia pastoris ) can be used as a platform for value-added chemicals using CO 2 as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. 13 C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO 2 as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L -1 , while balancing the expression of key genes and modifying screening conditions led to 2 g L -1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO 2 uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO 2 , to support sustainability of industrial bioprocesses.

Plants and green algae have a low pH-inducible mechanism in photosystem II (PSII) that dissipates excess light energy, measured as the nonphotochemical quenching of chlorophyll fluorescence (qE). Recently, nonphotochemical quenching 4 (npq4), a mutant strain of the green alga Chlamydomonas reinhardtii that is qE-deficient and lacks the light-harvesting complex stress-related protein 3 (LHCSR3), was reported [Peers G, et al. (2009) Nature 462(7272):518-521]. Here, applying a newly established procedure, we isolated the PSII supercomplex and its associated light-harvesting proteins from both WT C. reinhardtii and the npq4 mutant grown in either low light (LL) or high light (HL). LHCSR3 was present in the PSII supercomplex from the HL-grown WT, but not in the supercomplex from the LL-grown WT or mutant. The purified PSII supercomplex containing LHCSR3 exhibited a normal fluorescence lifetime at a neutral pH (7.5) by single-photon counting analysis, but a significantly shorter lifetime at pH 5.5, which mimics the acidified lumen of the thylakoid membranes in HL-exposed chloroplasts. The switch from light-harvesting mode to energy-dissipating mode observed in the LHCSR3-containing PSII supercomplex was sensitive to dicyclohexylcarbodiimide, a protein-modifying agent specific to protonatable amino acid residues. We conclude that the PSII-LHCII-LHCSR3 supercomplex formed in the HL-grown C. reinhardtii cells is capable of energy dissipation on protonation of LHCSR3. [ABSTRACT FROM AUTHOR]

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity. [ABSTRACT FROM AUTHOR]

SNPs in the first intron of FTO (fat mass and obesity associated) are strongly associated with human obesity. While it is not yet formally established that this effect is mediated through the actions of the FTO protein itself, loss of function mutations in FTO or its murine homologue Fto result in severe growth retardation, and mice globally overexpressing FTO are obese. The mechanisms through which FTO influences growth and body composition are unknown. We describe a role for FTO in the coupling of amino acid levels to mammalian target of rapamycin complex 1 signaling. These findings suggest that FTO may influence body composition through playing a role in cellular nutrient sensing. [ABSTRACT FROM AUTHOR]

Despite the importance of Mg2+ for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg2+ transport. However, the key questions-such as, the ion selectivity, the transport pathway, and the gating mechanism-have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg2+ during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg2+ to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs. [ABSTRACT FROM AUTHOR]

Large conductance, voltage- and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2x increase in vascular myocyte BK activity (EC50 = 45 μM), an action that requires β1 but not other (β2-β4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK α- (cbv1) and β1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK β1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+rβ1T165A,T169A channels were LC-unresponsive. However, cbv1 + rβ1T165A and cbv1 + rβ1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rβ1T169A was LC-insensitive, with rβ1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rβ1L172A, and cbv1 + rβ1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid α-hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for LC action. Therefore, β1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK β1, our study provides a structural basis for advancing β1 subunit-specific pharmacology of BK channels. [ABSTRACT FROM AUTHOR]

Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G. [ABSTRACT FROM AUTHOR]

Treatment of an achiral molecular ladder of C2h symmetry composed of five edge-sharing cyclobutane rings, or a [5]-ladderane, with acid results in cis- to trans-isomerization of end pyridyl groups. Solution NMR spectroscopy and quantum chemical calculations support the isomerization to generate two diastereomers. The NMR data, however, could not lead to unambiguous configurational assignments of the two isomers. Single-crystal X-ray diffraction was employed to determine each configuration. One isomer readily crystallized as a pure form and X-ray diffraction revealed the molecule as being achiral based on Ci symmetry. The second isomer resisted crystallization under a variety of conditions. Consequently, a strategy based on a cocrystallization was developed to generate single crystals of the second isomer. Cocrystallization of the isomer with a carboxylic acid readily afforded single crystals that confirmed a chiral ladderane based on C2 symmetry. The chiral ladderane and acid self-assembled to generate a five-component hydrogen-bonded complex that packs to form large solvent-filled homochiral channels of nanometer-scale dimensions. Whereas cocrystallizations are frequently applied to structure determinations of proteins, our study represents the first application of a cocrystallization to confirm the relative configuration of a small-molecule diastereomer generated in a solution-phase organic synthesis. [ABSTRACT FROM AUTHOR]

Pathogen-specific responses are characterized by preferred profiles of peptide+class I MHC (pMHCI) glycoprotein-specific T-cell receptor (TCR) Variable (V)-region use. How TCRV-region bias impacts TCRαβ heterodimer selection and resultant diversity is unclear. The DbPA224-specific TCR repertoire in influenza A virus-infected C57BL/6J (B6) mice exhibits a preferred TCRV-region bias toward the TRBV29 gene segment and an optimal complementarity determining region (CDR3) β-length of 6 aa. Despite these restrictions, DbPA224-specific BV29+ T cells use a wide array of unique CDR3β sequences. Structural characterization of a single, TRBV29+DbPA224-specific TCRαβ-pMHCI complex demonstrated that CDR3α amino acid side chains made specific peptide interactions, but the CDR3β main chain exclusively contacted peptides. Thus, length but not amino acid sequence was key for recognition and flexibility in Vβ-region use. In support of this hypothesis, retrovirus expression of the DbPA224-specific TCRVα-chain was used to constrain pairing within a naive/immune epitope-specific repertoire. The retrogenic TCRVα paired with a diversity of CDR3βs in the context of a preferred TCRVβ spectrum. Overall, these data provide an explanation for the combination of TCRV region bias and diversity within selected repertoires, even as they maintain exquisite pMHCI specificity. [ABSTRACT FROM AUTHOR]

Ca2+-activated Cl- channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca2+ has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca2+ or by voltage in the absence of Ca2+, but Ca2+- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca2+ and voltage sensing. Deleting 448EAVK in the first intracellular loop dramatically decreases apparent Ca2+ affinity. In contrast, mutating the adjacent amino acids 444EEEE abolishes intrinsic voltage dependence without altering the apparent Ca2+affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca2+ was facilitated by anions with high permeability or by an increase in [Cl-]e. Our data show that the transition between closed and open states is governed by Ca2+ in a voltage-dependent manner and suggest that anions allosterically modulate Ca2+-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic. [ABSTRACT FROM AUTHOR]

Prenylation is the posttranslational modification of a carboxyl-terminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells. [ABSTRACT FROM AUTHOR]

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders. [ABSTRACT FROM AUTHOR]

Ataxia with oculomotor apraxia 1 is caused by mutation in the APTX gene, which encodes the DNA strand-break repair protein aprataxin. Aprataxin exhibits homology to the histidine triad superfamily of nucleotide hydrolases and transferases and removes 5'-adenylate groups from DNA that arise from aborted ligation reactions. We report herein that aprataxin localizes to mitochondria in human cells and we identify an N-terminal amino acid sequence that targets certain isoforms of the protein to this intracellular compartment. We also show that transcripts encoding this unique N-terminal stretch are expressed in the human brain, with highest production in the cerebellum. Depletion of aprataxin in human SH-SY5Y neuroblastoma cells and primary skeletal muscle myoblasts results in mitochondrial dysfunction, which is revealed by reduced citrate synthase activity and mtDNA copy number. Moreover, mtDNA, not nuclear DNA, was found to have higher levels of background DNA damage on aprataxin knockdown, suggesting a direct role for the enzyme in mtDNA processing. [ABSTRACT FROM AUTHOR]

Voltage gating of hyperpolarization-activated cation (HCN) channels is potentiated by direct binding of cAMP to a cytoplasmic CAMP-sensing domain (CSD). When unliganded, the CSD inhibits hyperpolarization-dependent opening of the HCN channel gate; cAMP binding relieves this autoinhibition so that opening becomes more favorable thermodynamically. This autoinhibition-relief me- chanism is conserved with that of several other cyclic nucleotide receptors using the same ligand-binding fold. Besides its thermo- dynamic effect, cAMP also modulates the depolarization-depen- dent deactivation rate by kinetically trappingchannels in an open state. Here we report studies of strong open-state trapping in an HCN channel showing that the well-established autoinhibition- relief model is insufficient. Whereas deletion of the CSD mimics the thermodynamic open-state stabilization usually associated with CAMP binding. CSD deletion removes rather than mimics the kinetic effect of strong open-state trapping. Substitution of different CSD sequences leads to variation of the degree of open-state trapping in the liganded channel but not in the unliganded channel. CSDdependent open-state trapping is observed during a voltage- dependent deactivation pathway, specific to the secondary open state that is formed by mode shift after prolonged hyperpolarization activation. This hysteretic activation-deactivation cycle is preserved by CSD substitution, but the change in deactivation kinetics of the liganded channel resulting from CSD substitution is not correlated with the change in autoinhibition properties. Thus the liganded and the unhiganded forms of the CSD respectively provide the structural detejminants for open-state trapping and autoinhibition, such that two distinct mechanisms for cAMP regulation can operate in one receptor. [ABSTRACT FROM AUTHOR]

Phosphoeno!pyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae, and vascular plants, and is believed to be cytosolic. Here we show that rice (Oryza sativa L) has a plant-type PEPC, Osppc4, that is targeted to the chloroplast. Osppc4 was expressed in all organs tested and showed high expression in the leaves. Its expression in the leaves was confined to mesophyll cells, and Osppc4 accounted for approximately one-third of total PEPC protein in the leaf blade. Recombinant Osppc4 was active in the PEPC reaction, showing Vmax comparable to cytosolic isozymes. Knockdown of Osppc4 expression by the RNAi technique resulted in stunting at the vegetative stage, which was much more marked when rice plants were grown with ammonium than with nitrate as the nitrogen source. Comparison of leaf metabolomes of ammonium-grown plants suggested that the knockdown suppressed ammonium assimilation and subsequent amino acid synthesis by reducing levels of organic acids, which are carbon skeleton donors for these processes. We also identified the chloroplastic PEPC gene in other Oiyza species, all of which are adapted to waterlogged soil where the major nitrogen source is ammonium. This suggests that, in addition to glycolysis, the genus Oryza has a unique route to provide organic acids for ammonium assimilation that involves a chloroplastic PEPC, and that this route is crucial for growth with ammonium. This work provides evidence for diversity of primary ammonium assimilation in the leaves of vascular plants. [ABSTRACT FROM AUTHOR]

The PII protein is a signal integrator involved in the regulation of nitrogen metabolism in bacteria and plants. Upon sensing of cellular carbon and energy availability, PII conveys the signal by interacting with target proteins, thereby modulating their biological activity. Plant PII is located to plastids; therefore, to identify new PII target proteins, PII-affinity chromatography of soluble extracts from Arabidopsis leaf chloroplasts was performed. Several proteins were retained only when Mg-ATP was present in the binding medium and they were specifically released from the resin by application of a 2-oxoglutarate-containing elution buffer. Mass spectroscopy of SDS/PAGE-resolved protein bands identified the biotin carboxyl carrier protein subunits of the plastidial acetyl-CoA carboxylase (ACCase) and three other proteins containing a similar biotinllipoyl-binding motif as putative PII targets. ACCase is a key enzyme initiating the synthesis of fatty acids in plastids. In in vitro reconstituted assays supplemented with exogenous ATP, recombinant Arabidopsis PII inhibited chloroplastic ACCase activity, and this was completely reversed in the presence of 2-oxoglutarate, pyruvate, or oxaloacetate. The inhibitory effect was PII-dose-dependent and appeared to be PII-specific because ACCase activity was not altered in the presence of other tested proteins. PII decreased the Vmax of the ACCase reaction without altering the Km for acetyl-CoA. These data show that PII function has evolved between bacterial and plant systems to control the carbon metabolism pathway of fatty acid synthesis in plastids. [ABSTRACT FROM AUTHOR]

During metabolic evolution to improve succinate production in Escherichia coil strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The energy-conserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzymedominated CO2 fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsl. This glucose transport function was replaced by increased expression of the GaIP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GaIP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coil fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria. [ABSTRACT FROM AUTHOR]

It is demonstrated that, properly represented, the amino acid composition of protein sequences contains the information necessary to delineate the global properties of protein structure space. A numerical representation of amino acid sequence in terms of a set of property factors is used, and the values of those property factors are averaged over individual sequences and then over sets of sequences belonging to structurally defined groups. These sequence sets then can be viewed as points in a 10-dimensional space, and the organization of that space, determined only by sequence properties, is similar at both local and global scales to that of the space of protein structures determined previously. [ABSTRACT FROM AUTHOR]

One of the largest contributions to biologically available nitrogen comes from the reduction of N2 to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids. and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N2 fixation by Rhizobium leguminosarum by viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N2 fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N2 fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis. [ABSTRACT FROM AUTHOR]

Core genes comprising the ubiquitous backbone of bacterial genomes are not subject to frequent horizontal transfer and generally are not thought to contribute to the adaptive evolution of bacterial pathogens. We determined, however, that at least one-third and possibly more than one-half of the core genes in Escherichia coil genomes are targeted by repeated replacement substitutions in the same amino acid positionshotspot mutations. Occurrence of hotspot mutations is driven by positive selection, as their rate is significantly higher than expected by random chance alone, and neither intragenic recombination nor increased mutability can explain the observed patterns. Also, commensal E. coiistrains have a significantly lower frequency of mutated genes and mutations per genome than pathogenic strains. E. co/i strains causing extra-intestinal infections accumulate hotspot mutations at the highest rate, whereas the highest total number of mutated genes has been found among Shigeiia isolates, suggesting the pathoadaptive nature of such mutations. The vast majority of hotspot mutations are of recent evolutionary origin, implying short-term positive selection, where adaptive mutations emerge repeatedly but are not sustained in natural circulation for long. Such pattern of dynamics is consistent with source-sink model of virulence evolution. [ABSTRACT FROM AUTHOR]

Infectious tolerance describes the process of CD4+ regulatory T cells (Tregs) converting naïve T cells to become additional Tregs. We show that antigen-specific Tregs induce, within skin grafts and dendritic cells, the expression of enzymes that consume at least 5 different essential amino acids (EAAs). T cells fail to proliferate in response to antigen when any 1, or more, of these EAAs are limiting, which is associated with a reduced mammalian target of rapamycin (mTOR) signaling. Inhibition of the mTOR pathway by limiting EAAs, or by specific inhibitors, induces the Treg-specific transcription factor forkhead box P3. which depends on both T cell receptor activation and synergy with TGF-β. [ABSTRACT FROM AUTHOR]

We describe a general application of the nonsense suppression methodology for unnatural amino acid incorporation to probe drug-receptor interactions in functional G protein-coupled receptors (GPCRs), evaluating the binding sites of both the M2 muscarinic acetylcholine receptor and the D2 dopamine receptor. Receptors were expressed in Xenopus oocytes. and activation of a G protein-coupled, inward-rectifying K+ channel (GIRK) provided, after optimization of conditions, a quantitative readout of receptor function. A number of aromatic amino acids thought to be near the agonist-binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at W6.48 of the D2 receptor establishes a cation-π interaction between the agonist dopamine and W6.48, suggesting a reorientation of W6.48 on agonist binding, consistent with proposed "rotamer switch" models. Interestingly, no comparable cation-π interaction was found at the aligning residue in the M2 receptor. [ABSTRACT FROM AUTHOR]

Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. These experiments offer empirical lessons about how proteins evolve in the face of clearly-defined laboratory selection pressures. Directed evolution has revealed that single amino acid mutations can enhance properties such as catalytic activity or stability and that adaptation can often occur through pathways consisting of sequential beneficial mutations. When there are no single mutations that improve a particular protein property experiments always find a wealth of mutations that are neutral with respect to the laboratory-defined measure of fitness. These neutral mutations can open new adaptive pathways by at least 2 different mechanisms. Functionally- neutral mutations can enhance a protein's stability, thereby increasing its tolerance for subsequent functionally beneficial but destabilizing mutations. They can also lead to changes in "promiscuous" functions that are not currently under selective pressure, but can subsequently become the starting points for the adaptive evolution of new functions. These lessons about the coupling between adaptive and neutral protein evolution in the laboratory offer insight into the evolution of proteins in nature. [ABSTRACT FROM AUTHOR]

Alanyl-tRNA synthetase (AIaRS) specifically recognizes the major identity determinant, the G3:U70 base pair, in the acceptor stem of tRNAAIa by both the tRNA-recognition and editing domains. In this study, we solved the crystal structures of 2 halves of Archaeoglobus fulgidus AIaRS: AIaRS-ΔC, comprising the aminoacylation, tRNA-recognition, and editing domains, and AIaRS-C, comprising the dimerization domain. The aminoacylation/tRNA-recognition domains contain an insertion incompatible with the class-specific tRNA-binding mode. The editing domain is fixed tightly via hydrophobic interactions to the aminoacylation/tRNA-recognition domains, on the side opposite from that in threonyl-tRNA synthetase. A groove formed between the aminoacylation/tRNA-recognition domains and the editing domain appears to be an alternative tRNA-binding site, which might be used for the aminoacylation and/or editing reactions. Actually, the amino acid residues required for the G3:U70 recognition are mapped in this groove. The dimerization domain consists of helical and globular subdomains. The helical subdomain mediates dimerization by forming a helix-loop-helix zipper. The globular subdomain, which is important for the aminoacylation and editing activities, has a positively-charged face suitable for tRNA binding. [ABSTRACT FROM AUTHOR]

Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (≈3,000 s-1), with the extrapolated folding rate constant at ≈20 °C in physiological conditions reaching 20,000 s-1. By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant ϕ-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrPC conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact (m value analysis gives a βt value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence. [ABSTRACT FROM AUTHOR]

The distribution and enantiomeric composition of the 5-carbon (C5) amino acids found in CI-, CM-, and CR-type carbonaceous meteorites were investigated by using liquid chromatography fluorescence detection/TOF-MS coupled with o-phthaldialdehyde/N-acetyl-l-cysteine derivatization. A large l-enantiomeric excess (ee) of the a-methyl amino acid isovaline was found in the CM meteorite Murchison (∟ee = 18.5 ± 2.6%) and the CI meteorite Orgueil (∟ee = 15.2 ± 4.0%). The measured value for Murchison is the largest enantiomeric excess in any meteorite reported to date, and the Orgueil measurement of an isovaline excess has not been reported previously for this or any CI meteorite. The ∟-isovaline enrichments in these two carbonaceous meteorites cannot be the result of interference from other C5 amino acid isomers present in the samples, analytical biases, or terrestrial amino acid contamination. We observed no ∟-isovaline enrichment for the most primitive unaltered Antarctic CR meteorites EET 92042 and QUE 99177. These results are inconsistent with UV circularly polarized light as the primary mechanism for ∟-isovaline enrichment and indicate that amplification of a small initial isovaline asymmetry in Murchison and Orgueil occurred during an extended aqueous alteration phase on the meteorite parent bodies. The large asymmetry in isovaline and other α-dialkyl amino acids found in altered CI and CM meteorites suggests that amino acids delivered by asteroids, comets, and their fragments would have biased the Earth's prebiotic organic inventory with left-handed molecules before the origin of life. [ABSTRACT FROM AUTHOR]

Aminoacyl-tRNA synthetases (aaRSs) join amino acids to 1 of 2 terminal hydroxyl groups of their cognate tRNAs, thereby contributing to the overall fidelity of protein synthesis. In class II histidyl-tRNA synthetase (HisRS) the nonbridging Sp-oxygen of the adenylate is a potential general base for aminoacyl transfer. To test for conservation of this mechanism in other aaRSs and the role of terminal hydroxyls of tRNA in aminoacyl transfer, we investigated the class II Escherichia coli threonyl-tRNA synthetase (ThrRS). As with other class II aaRSs, the rate-determining step for ThrRS is amino acid activation. In ThrRS, however, the 2'-OH of A76 of tRNAThr and a conserved active-site histidine (His-309) collaborate to catalyze aminoacyl transfer by a mechanism distinct from HisRS. Conserved residues in the ThrRS active site were replaced with alanine, and then the resulting mutant proteins were analyzed by steady-state and rapid kinetics. Nearly all mutants preferentially affected the amino acid activation step, with only a modest effect on aminoacyl transfer. By contrast, H309A ThrRS decreased transfer 242-fold and imposed a kinetic block to CCA accommodation. His-309 hydrogen bonds to the 2′-OH of A76, and substitution of the latter by hydrogen or fluorine decreased aminoacyl transfer by 763- and 94-fold, respectively. The proton relay mechanism suggested by these data to promote aminoacylation is reminiscent of the NAD+-dependent mechanisms of alcohol dehydrogenases and sirtuins and the RNA-mediated catalysis of the ribosomal peptidyl transferase center. [ABSTRACT FROM AUTHOR]

A recently determined set of 20 NMR-derived conformations of a 48-residue all-α-helical protein, (PDB ID code 2JVD), is validated here by comparing the observed 13Cα chemical shifts with those computed at the density functional level of theory. In addition, a recently introduced physics-based method, aimed at determining protein structures by using NOE-derived distance constraints together with observed and computed 13Cα chemical shifts, was applied to determine a new set of 10 conformations, (Set-bt), as a blind test for the same protein. A cross-validation of these two sets of conformations in terms of the agreement between computed and observed 13Cα chemical shifts, several stereochemical quality factors, and some NMR quality assessment scores reveals the good quality of both sets of structures. We also carried out an analysis of the agreement between the observed and computed 13Cα chemical shifts for a slightly longer construct of the protein solved by x-ray crystallography at 2.0-Å resolution (PDB ID code 3BHP) with an identical amino acid residue sequence to the 2JVD structure for the first 46 residues. Our results reveal that both of the NMR-derived sets, namely 2JVD and Set-bt, are somewhat better representations of the observed 13Cα chemical shifts in solution than the 3BHP crystal structure. In addition, the 13Cα-based validation analysis appears to be more sensitive to subtle structural differences across the three sets of structures than any other NMR quality-assessment scores used here, and, although it is computationally intensive, this analysis has potential value as a standard procedure to determine, refine, and validate protein structures. [ABSTRACT FROM AUTHOR]

Here, we compare the distributions of main chain (ΦΨ) angles (i.e., Ramachandran maps) of the 20 naturally occurring amino acids in three contexts: (i) molecular dynamics (MD) simulations of Gly-Gly-X-Gly-Gly pentapeptides in water at 298 K with exhaustive sampling, where X = the amino acid in question; (ii) 188 independent protein simulations in water at 298 K from our Dynameomics Project; and (iii) static crystal and NMR structures from the Protein Data Bank. The GGXGG peptide series is often used as a model of the unstructured denatured state of proteins. The sampling in the peptide MD simulations is neither random nor uniform. Instead, individual amino acids show preferences for particular conformations, but the peptide is dynamic, and interconversion between conformers is facile. For a given amino acid, the (ΦΨ) distributions in the protein simulations and the Protein Data Bank are very similar and often distinct from those in the peptide simulations. Comparison between the peptide and protein simulations shows that packing constraints, solvation. and the tendency for particular amino acids to be used for specific structural motifs can overwhelm the "intrinsic propensities" of amino acids for particular (4,'I') conformations. We also compare our helical propensities with experimental consensus values using the host-guest method, which appear to be determined largely by context and not necessarily the intrinsic conformational propensities of the guest residues. These simulations represent an improved coil library free from contextual effects to better model intrinsic conformational propensities and provide a detailed view of conformations making up the "random coil" state. [ABSTRACT FROM AUTHOR]

During translation, usually only one in ≈400 misincorporations affects the function of a nascent protein, because only chemically similar near-cognate amino acids are misincorporated in place of the cognate one. The deleterious misincorporation of a chemically dissimilar noncognate amino acid during the selection process is precluded by the presence of a tRNA at the ribosomal E-site. However, the selection of first aminoacyl-tRNA, directly after initiation, occurs without an occupied E-site, i.e., when only the P-site is filled with the initiator tRNA and thus should be highly error-prone. Here, we show how bacterial ribosomes have solved this accuracy problem: In the absence of a Shine-Dalgarno (SD) sequence, the first decoding step at the A-site after initiation is extremely error-prone, even resulting in the significant incorporation of noncognate amino acids. In contrast, when a SD sequence is present, the incorporation of noncognate amino acids is not observed. This is precisely the effect that the presence of a cognate tRNA at the E-site has during the elongation phase. These findings suggest that during the initiation phase, the SD interaction functionally compensates for the lack of codon-anticodon interaction at the E-site by reducing the misincorporation of near-cognate amino acids and prevents noncognate misincorporation. [ABSTRACT FROM AUTHOR]

The proprotein convertase PC516 cleaves protein precursors after basic amino acids and is essential for implantation in CD11129/Sv/ C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdfll-related phenotypes such as altered an- teroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdfll-independent pheno- types, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC516 and Gdf 11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PCS/6 for Gdf 11 essentially resides in the presence of a P1' Asn in the RSRR ~ N cleavage motif. This work identifies Gdf 11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase. [ABSTRACT FROM AUTHOR]

Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K+ current lK1 and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac lK1 The voltage- and K+-dependence of chioroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chioroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block lK1 even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties. [ABSTRACT FROM AUTHOR]

The recombinant HIV-1 Tat protein contains a small region corresponding to residues 47YGRKKRRQRR57R, which is capable of translocating cargoes of different moIecuIar sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner. The pathway that these peptides follow for entry into the cell has been the subject of strong controversy for the last decade. This peptide is highly basic and hydrophilic. Therefore, a central question that any candidate mechanism has to answer is how this highly hydrophilic peptide is able to cross the hydrophobic barrier imposed by the cell membrane. We propose a mechanism for the spontaneous translocation of the Tat peptides across a lipid membrane. This mechanism involves strong interactions between the Tat peptides and the phosphate groups on both sides the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of the Tat peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically the arginine amino acids, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Indeed, mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions. [ABSTRACT FROM AUTHOR]

The chaperonin GroEL binds non-native polypeptides in an open ring via hydrophobic contacts and then, after ATP and GroES binding to the same ring as polypeptide, mediates productive folding in the now hydrophilic, encapsulated cis chamber. The nature of the folding reaction in the cis cavity remains poorly understood. In particular, it is unclear whether polypeptides take the same route to the native state in this cavity as they do when folding spontaneously free in solution. Here, we have addressed this question by using NMR measurements of the time course of acquisition of amide proton exchange protection of human dihydrofolate reductase (DHFR) during folding in the presence of methotrexate and ATP either free in solution or inside the stable cavity formed between a single ring variant of GroEL, SR1, and GroES. Recovery of DHFR refolded by the SR1/GroES-mediated reaction is 2-fold higher than in the spontaneous reaction. Nevertheless, DHFR folding was found to proceed by the same trajectories inside the cis folding chamber and free in solution. These observations are consistent with the description of the chaperonin chamber as an "Anfinsen cage" where polypeptide folding is determined solely by the amino acid sequence, as it is in solution. However, if misfolding occurs in the confinement of the chaperonin cavity, the polypeptide chain cannot undergo aggregation but rather finds its way back to a productive pathway in a manner that cannot be accomplished in solution, resulting in the observed high overall recovery. [ABSTRACT FROM AUTHOR]

During HIV-1 entry, the highly conserved gp41 N-trimer pocket region becomes transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (D- peptides) that bind the N-trimer pocket with high affinity and potently inhibit viral entry. We also report high-resolution crystal structures of two of these D-peptides in complex with a pocket mimic that suggest sources of their high potency. A trimeric version of one of these peptides is the most potent pocket-specific entry inhibitor yet reported by three orders of magnitude (1C50 = 250 pM). These results are the first demonstration that D-peptides can form specific and high-affinity interactions with natural protein targets and strengthen their promise as therapeutic agents. The D-peptides described here address limitations associated with current L-peptide entry inhibitors and are promising leads for the prevention and treatment of HIV/AIDS. [ABSTRACT FROM AUTHOR]