Nitrile regio-synthesis by Ni centers on a siliceous surface: implications in prebiotic chemistry.

By means of quantum chemistry (PBE0/def2-TZVPP; DLPNO-CCSD(T)/cc-pVTZ) and small, but reliable models of Polyhedral Oligomeric Silsesquioxanes (POSS), an array of astrochemically-relevant catalysis products, related to prebiotic and origin of life chemistry, has been theoretically explored. In this work, the heterogeneous phase hydrocyanation reaction of an unsaturated CC bond (propene) catalyzed by a Ni center complexed to a silica surface is analyzed. Of the two possible regioisomers, the branched iso-propyl-cyanide is thermodynamically and kinetically preferred over the linear n-propyl-cyanide (T = 200 K). The formation of nitriles based on a regioselective process has profound implications on prebiotic and origin of life chemistry, as well as deep connections to terrestrial surface chemistry and geochemistry.

Complex Organic Matter Synthesis on Siloxyl Radicals in the Presence of CO.

Heterogeneous phase astrochemistry plays an important role in the synthesis of complex organic matter (COM) as found on comets and rocky body surfaces like asteroids, planetoids, moons and planets. The proposed catalytic model is based on two assumptions: (a) siliceous rocks in both crystalline or amorphous states show surface-exposed defective centers such as siloxyl (Si-O•) radicals; (b) the second phase is represented by gas phase CO molecules, an abundant C1 building block found in space. By means of quantum chemistry; (DFT, PW6B95/def2-TZVPP); the surface of a siliceous rock in presence of CO is modeled by a simple POSS (polyhedral silsesquioxane) where a siloxyl (Si-O•) radical is present. Four CO molecules have been consecutively added to the Si-O• radical and to the nascent polymeric CO (pCO) chain. The first CO insertion shows no activation free energy with ΔG200K = -21.7 kcal/mol forming the SiO-CO• radical. The second and third CO insertions show Δ G 200 K ‡ ≤ 10.5 kcal/mol. Ring closure of the SiO-CO-CO• (oxalic anhydride) moiety as well as of the SiO-CO-CO-CO• system (di-cheto form of oxetane) are thermodynamically disfavored. The last CO insertion shows no free energy of activation resulting in the stable five member pCO ring, precursor to 1,4-epoxy-1,2,3-butanone. Hydrogenation reactions of the pCO have been considered on the SiO oxygen or on the carbons and oxygens of the pCO chains. The formation of the reactive aldehyde SiO-CHO on the siliceous surface is possible. In principle, the complete hydrogenation of the (CO)1-4 series results in the formation of methanol and polyols. Furthermore, all the SiO-pCO intermediates and the lactone 1,4-epoxy-1,2,3-butanone product in its radical form can be important building blocks in further polymerization reactions and/or open ring reactions with H (aldehydes, polyols) or CN (chetonitriles), resulting in highly reactive multi-functional compounds contributing to COM synthesis.

On the formation of phosphorous polycyclic aromatics hydrocarbons (PAPHs) in astrophysical environments.

The formation of phosphorous-containing polycyclic aromatic hydrocarbons (PAPHs) in astrophysical contexts is proposed and analyzed by means of computational methods [B3LYP-D3BJ/ma-def2-TZVPP, MP2-F12, CCSD-F12b and CCSD(T)-F12b levels of theory]. A "bottom-up" approach based on a radical-neutral reaction scheme between acetylene (C2H2) and the CP radical was used investigating: (a) the synthesis of the first PAPH (C5H5P) "phosphinine"; (b) PAPH growth by addition of C2H2 to the C5H4P radical; (c) PAPH synthesis by addition reactions of one CP radical and nC2H2 to a neutral PAH. Results show: (I) the formation of the phosphinine radical has a strong thermodynamic tendency (-133.3 kcal mol-1) and kinetic barriers ≤5.4 kcal mol-1; (II) PAPH growth by nC2H2 addition on the radical phosphinine easily and exothermically produces radicals (1a- or 1-phospha-naphtalenes with kinetic barriers ≤7.1 kcal mol-1 and reaction free energies ≤-102.5 kcal mol-1); (III) the addition of a single CP + nC2H2 to a neutral benzene generates a complex chemistry where the main product is 2-phospha-naphtalene; (IV) because of the CP radical character, its barrierless addition to a PAH produces a resonant stabilized PAPH, becoming excellent candidates for addition reactions with neutral or radical hydrocarbons and PAHs; (V) the same energy trend between all four levels of theory continues a well-calibrated computational protocol to analyze complex organic reactions with astrochemical interest using electronic structure theory.

Propylene Oxide Formation on a Silica Surface with Peroxo Defects: Implications in Astrochemistry.

The formation of the chiral molecule propylene oxide (CH3CHCH2O) recently detected in the interstellar medium (ISM) is proposed to take place on an amorphous silicate grain surface where peroxo defects are present. A computational analysis conducted at the DFT and MP2-F12 levels of theory on a neat amorphous silica model supports such a hypothesis resulting in (a) strong thermodynamic driving forces and low activation energies allowing the synthesis of CH3CHCH2O at low temperatures, (b) chemical defects on silica surfaces promoting heterogeneous catalysis of the increasing molecular complexity found in interstellar and circumstellar medium, and (c) chemical defects that have implications on understanding how processing phases modify the nature of the reactive groups on a silica surface affecting the surface catalytic activity.

H2 Formation on Cosmic Grain Siliceous Surfaces Grafted with Fe+ : A Silsesquioxanes-Based Computational Model.

Cosmic siliceous dust grains are involved in the synthesis of H2 in the inter-stellar medium. In this work, the dust grain siliceous surface is represented by a hydrogen Fe-metalla-silsesquioxane model of general formula: [Fe(H7 Si7 O12-n )(OH)n ]+ (n=0,1,2) where Fe+ behaves like a single-site heterogeneous catalyst grafted on a siliceous surface synthesizing H2 from H. A computational analysis is performed using two levels of theory (B3LYP-D3BJ and MP2-F12) to quantify the thermodynamic driving force of the reaction: [Fe-T7H7 ]+ +4H→[Fe-T7H7 (OH)2 ]+ +H2 . The general outcomes are: 1) H2 synthesis is thermodynamically strongly favored; 2) Fe-H / Fe-H2 barrier-less formation potential; 3) chemisorbed H-Fe leads to facile H2 synthesis at 20≤T≤100 K; 4) relative spin energetics and thermodynamic quantities between the B3LYP-D3BJ and MP2-F12 levels of theory are in qualitative agreement. The metalla-silsesquioxane model shows how Fe+ fixed on a siliceous surface can potentially catalyze H2 formation in space.

Astrochemistry of transition metals? The selected cases of [FeN](+), [FeNH](+) and [(CO)2FeN](+): pathways toward CH3NH2 and HNCO.

Transition metals (TMs) are proposed to play a role in astrophysical environments in both gas and solid state astrochemistry by co-determining the homogeneous/heterogeneous chemistry represented by the gas-gas and gas-dust grain interactions. Their chemistry is a function of temperature, radiation field and chemical composition/coordination sphere and as a consequence, dependent on the astrophysical object in which TMs are localized. Here five main categories of TM compounds are proposed and classified as: (a) pure bulk and clusters; (b) TM naked ions; (c) TM oxides/minerals or inorganic compounds; (d) TM-L (L = ligand) with L = (σ and/or π)-donor/acceptor species like H/H2, N/N2, CO, and H2O and (e) TM-organoligands such as Cp, PAH, and R1=˙=˙=R2. Each of the classes is correlated to their possible localization within astrophysical objects. Because of this variety coupled with their ability to modulate reactivity and regio/enantioselectivity by ligand sphere composition, TM compounds can introduce a fine organic synthesis in astrochemistry. For the selection of small TM parental compounds to be analyzed as first examples, by constraining the TMs and the second element/molecule on the basis of their cosmic abundance and mutual reactivity, Fe atoms coupled with N and CO are studied by developing the chemistry of [FeN](+), [FeNH](+) and [(CO)2FeN](+). These molecules, due to their ability to perform C-C and C-H bond activation, are able to open the pathway toward nitrogenation/amination and carbonylation of organic substrates. By considering the simplest organic substrate CH4, the parental reaction schemes (gas phase, T = 30 K): (I) [FeN](+) + CH4 + H → [Fe](+) + H3C-NH2; (II) [FeNH](+) + CH4 → [Fe](+) + H3C-NH2 and (III) [(CO)2FeN](+) + H → [FeCO](+) + HNCO are analyzed by theoretical methods (B2PLYP double hybrid functional/TZVPPP basis set). All reactions are thermodynamically favored and first step transition states can follow a minimal energy path by spin crossing, while H extraction in reaction II shows very high activation energies. The need to overcome high activation energy barriers underlines the importance of molecular activation by radiation and particle collision. TM chemistry is expected to contribute to the known synthesis of organic compounds in space leading towards a new direction in the astrochemistry field whose qualitative (type of compounds) and quantitative contributions must be unraveled.

Enhanced EGFP fluorescence emission in presence of PEG aqueous solutions and PIB1000-PEG6000-PIB1000 copolymer vesicles.

An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1:10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

Structural and dynamical analysis of an engineered FhuA channel protein embedded into a lipid bilayer or a detergent belt.

Engineered channel proteins are promising nano-components with applications in nanodelivery and nanoreactors technology. Because few of the engineered channel proteins have been crystallized, solution studies based on Neutron Scattering, Circular Dichroism and NMR play a major role. Consequently, the understanding of membrane proteins dynamics in water/detergent solutions or when embedded in a lipid membrane, can clarify how the environment affects protein behavior. In this study, molecular dynamics simulations of the FhuA Escherichia coli outer membrane channel protein and its engineered FhuA Δ1-159 variant have been performed in two different environments: a DNPC (1,2-dinervonyl-sn-glycero-3-phosphocholine) lipid bilayer and a water/OES (N-octyl-2-hydroxyethyl sulfoxide) detergent solution. Furthermore the FhuA Δ1-159 variant has been simulated in the open and closed states, the last induced by the presence of six 3-(2-pyridyldithio)-propionic-acid in the channel inner core. Differences in protein structural and dynamical behavior between the two environments have been found. Considering the FhuA protein characterized by an elliptical-cylindrical symmetry: (a) neither variations on the secondary structure nor axial deformation have been observed in any of the systems; (b) the ellipticity of the channel section (open state) and its fluctuations are enhanced in presence of water/OES, while diminished or suppressed in the DNPC bilayer; (c) the insertion of hydrophobic pyridyl groups into the FhuA Δ1-159 channel (closed state) induces a higher ellipticity in water/OES solution, while shifting to a circular section in the DNPC membrane; (d) the cork domain represented by the first 159 amino acids does not play a major role for protein stability.

Polymersome surface decoration by an EGFP fusion protein employing Cecropin A as peptide "anchor".

Polymer based nanocompartments have potential applications in synthetic biology, medicine (drug release) and industrial biotechnology (chiral nanoreactors, multistep syntheses, selective product recovery). A step towards the aforementioned goals is the polymer membrane functionalization through covalent bonding of chemical anchors or insertion of proteins/peptides, to obtain specific properties like recognition, catalytic activity and facilitated diffusion, mimicking the complexity of a biological membrane. The use of genetic engineering techniques widens the possible applications of peptides and proteins specifically designed for polymer membrane interactions. A fusion protein (CecEGFP) based on the antibacterial peptide Cecropin A and the EGFP (Enhanced Green Fluorescent Protein) was designed, expressed and biophysically characterized. CecEGFP interaction with the tri-block copolymer PIB-PEG-PIB (PIB=polyisobutylene, PEG=polyethylene glycol) based polymersome membrane was analyzed by circular dichroism as well as EGFP and Trp fluorescence measurements. Results proved that Cecropin A is usable as a "membrane surface anchor" for water soluble proteins, as it inserts into the polymer membrane. The aim and novelty of this study is within the design of fusion proteins specifically developed for polymer membrane interactions. The use of amphiphilic Cecropin A "anchoring" water soluble proteins to the polymersome surface, avoids chemical coupling between polymers and proteins.

Engineering of an E. coli outer membrane protein FhuA with increased channel diameter.

BACKGROUND: Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. RESULTS: The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal ß-strands, resulting in variant FhuA Δ1-159 Exp. The total number of ß-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high ß-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. CONCLUSION: In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

Effect of Na+, Mg2+, and Zn2+ chlorides on the structural and thermodynamic properties of water/n-heptane interfaces.

The effect on the structural and thermodynamic properties in water/n-heptane interfaces on addition of NaCl, MgCl(2), and ZnCl(2) has been examined through five independent 100-ns molecular dynamics simulations. Results indicate that the interfacial thickness within the framework of the capillary-wave model decreases on addition of electrolytes in the order Na(+) < Mg(2+) < Zn(2+), whereas the interfacial tension increases in the same order. Ionic density profiles and self-diffusion coefficients are strongly influenced by the strength of the first hydration shell, which varies in the order Na(+) < Mg(2+) < Zn(2+). On the other hand, the Cl(-) behavior, that is, diffusion and solvation sphere, is influenced by its counterion. Accordingly, cations are strongly expelled from the interface, which is especially remarkable for the small divalent cations. This fact alters the water geometry near the interface and in a lesser extent n-heptane order and number of hydrogen bonds per water molecule close to the interface.

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile.

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80°C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135±6% and 193±8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50°C and about 45°C respectively, while 65°C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.

Engineering of the E. coli outer membrane protein FhuA to overcome the hydrophobic mismatch in thick polymeric membranes.

BACKGROUND: Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. RESULTS: To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 ß-sheets, prior to the more regular periplasmatic ß-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the ß-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB(1000)-PEG(6000)-PIB(1000) (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB(1000)-PEG(6000)-PIB(1000) membrane. Furthermore labeling of the Lys-NH(2) groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. CONCLUSION: Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.