Ligand migration in nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana

AHb1 is a hexacoordinated type 1 nonsymbiotic hemoglobin recently discovered in Arabidopsis thaliana. To gain insight into the ligand migration inside the protein, we studied the CO rebinding kinetics of AHb1 encapsulated in silica gels, in the presence of glycerol. The CO rebinding kinetics after nanosecond laser flash photolysis exhibits complex ligand migration patterns, consistent with the existence of discrete docking sites in which ligands can temporarily be stored before rebinding to the heme at different times. This finding may be of relevance to the physiological NO dioxygenase activity of this protein, which requires sequential binding of two substrates, NO and O2, to the heme.     

Constant pH Molecular Dynamics in Explicit Solvent with λ-Dynamics

pH is an important parameter in condensed-phase systems, because it determines the protonation state of titratable groups and thus influences the structure, dynamics, and function of molecules in solution. In most force field simulation protocols, however, the protonation state of a system (rather than its pH) is kept fixed and cannot adapt to changes of the local environment. Here, we present a method, implemented within the MD package GROMACS, for constant pH molecular dynamics simulations in explicit solvent that is based on the λ-dynamics approach. In the latter, the dynamics of the titration coordinate λ, which interpolates between the protonated and deprotonated states, is driven by generalized forces between the protonated and deprotonated states. The hydration free energy, as a function of pH, is included to facilitate constant pH simulations. The protonation states of titratable groups are allowed to change dynamically during a simulation, thus reproducing average protonation probabilities at a certain pH. The accuracy of the method is tested against titration curves of single amino acids and a dipeptide in explicit solvent.     

A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection

In this work, the characterisation and the optimisation of hybridisation assays based on a novel, rapid and sensitive micro-analytical, gravity-driven, flow device is reported. This device combines a special chip containing eight polymer microchannels, with a portable, computer-controlled instrument. The device is used as a platform for affinity experiments using oligonucleotide-modified paramagnetic particles. In our approach, both hybridisation and labelling events are performed on streptavidin-coated paramagnetic microparticles functionalized with a biotinylated capture probe. Modified particles, introduced in the microchannel inlet of the chip, accumulate near the electrode surface by virtue of a magnetic holder. After hybridisation with the complementary sequence, the hybrid is labelled with an alkaline phosphatase conjugate. The electrochemical substrate for alkaline phosphatase revelation is p-aminophenyl phosphate. Solutions and reagents are sequentially passed through the microchannels, until enzyme substrate is added for in situ signal detection. Upon readout, the magnet array is flipped away, beads are removed by addition of regeneration buffer, and the so-regenerated chip is ready for further analysis. This protocol has been applied to the analytical detection of specific DNA sequences of Legionella pneumophila, with an RSD=8.5% and a detection limit of 0.33 nM.     

β-Nitroacrylates and silyl enol ethers as key starting materials for the synthesis of polyfunctionalized β-nitro esters and 1,2-oxazine-2-oxides

The reaction of silyl enol ethers with β-nitroacrylates, in the presence of tetrabutyl ammonium fluoride as catalyst, allows the formation of polyfunctionalized β-nitro esters, or hexahydro-4H-benzoxazine-2-oxides, depending on the nature of the starting silyl enol ethers.     

Linear polyisophthalamides from the trifunctional monomer 5-hydroxyisophthalic acid. A convenient approach towards functionalized aromatic polymers

Wholly aromatic polyisophthalamides (PIPAs) containing free hydroxylic groups were prepared from 5-hydroxyisophthalic acid and sulfonyldianiline by the condensation phosphorylation method, using triphenylphosphite as a condensing agent, in the presence of pyridine. Linear polymers were attained in high yield and high molecular weight, and the properties of the polymers were investigated in comparison with homologous polyisophthalamides without OH groups. The presence of OH groups did not much modified the solubility of hydroxyl-containing polyisophthalamides as they behaved against common solvents in a similar manner as that of reference polyisophthalamides. However, hydroxyl side groups greatly enhanced the water affinity of polyisophthalamides, which showed up to 16% water uptake in 98% relative humidity, and contact angles about 10° smaller than those of classical PIPAs. The presence of free OH groups slightly diminished the thermal resistance, as evaluated by TGA, but the glass transition temperatures were higher by 20–30 °C, with a maximum of 345 °C. The mechanical properties of hydroxyl-containing polyisophthalamides were reasonably acceptable, with modulus around 2000 MPa and tensile strength up to 60 MPa.     

Dynamic hydrogen-bonding network in the distal pocket of the nitrosyl complex of Pseudomonas aeruginosa cd1 nitrite reductase

cd(1) nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d(1)-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d(1)-heme to avoid enzyme inhibition by the formation of ferrous-nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d(1)-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d(1)-heme NO complex from Pseudomonas aeruginosa cd(1) NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the "distal" side of the heme (Tyr(10), His(327), and His(369)), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron-nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d(1)-heme complex of cd(1) NIR forms H-bonds with Tyr(10) and His(369), whereas the second conserved histidine, His(327), appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr(10) is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His(369) is closer to the NO, whereas mutation of both distal histidines displaces Tyr(10), removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.     

Electronic and molecular structures of the members of the electron transfer series [Cr(tbpy)3]n (n = 3+, 2+, 1+, 0): an X-ray absorption spectroscopic and density functional theoretical study

The electron transfer series of complexes [Cr((t)bpy)(3)](n)(PF(6))(n) (n = 3+, 2+, 1+, 0 (1-4)) has been synthesized and the molecular structures of 1, 2, and 3 have been determined by single-crystal X-ray crystallography; the structure of 4 has been investigated using extended X-ray absorption fine structure (EXAFS) analysis. Magnetic susceptibility measurements (4-300 K) established an S = 3/2 ground state for 1, an S = 1 ground state for 2, an S = 1/2 ground state for 3, and an S = 0 ground state for 4. The electrochemistry of this series in CH(3)CN solution exhibits three reversible one-electron transfer waves. UV-vis/NIR spectra and Cr K-edge X-ray absorption spectra (XAS) are reported. The same experimental techniques have been applied for [Cr(III)(tacn)(2)]Br(3)·5H(2)O (5) and [Cr(II)(tacn)(2)]Cl(2) (6), which possess an S = 3/2 and an S = 2 ground state, respectively (tacn = 1,4,7-triazacyclononane, a tridentate, pure σ-donor ligand). The Cr K-edge XAS spectra of the corresponding complexes K(4)[Cr(II)(CN)(6)]·10H(2)O (S = 1) (7) and K(3)[Cr(III)(CN)(6)] (S = 3/2) (8) have also been recorded. All complexes have been studied computationally with density functional theory (DFT) using the B3LYP functional. The molecular and electronic structures of the anionic members of the series [Cr(bpy)(3)](1-,2-,3-) have also been calculated. It is unequivocally shown that all members of the electron transfer series 1-4 and [Cr(bpy)(3)](n) (n = 3+, 2+, 1+, 0, 1-, 2, 3-) possess a central Cr(III) ion ((t(2g))(3), S = 3/2). The three N,N'-coordinated neutral (bpy(0)) ligands in the trication 1 and [Cr(III)(bpy)(3)](3+) are one-electron reduced in a stepwise fashion to localized one, two, and three π-radical anions (bpy(?))(1-) in the dicationic, monocationic, and neutral species, respectively. Complexes 2 and [Cr(bpy)(3)](2+) cannot be described as low-spin Cr(II) species; they are in fact best described as [Cr(III)((t)bpy(?))((t)bpy(0))(2)](2+) and [Cr(III)(bpy(?))(bpy(0))(2)](2+) species. Further one-electron reductions yield one, two, and three diamagnetic (bpy(2-))(2-) dianions in the mono-, di-, and trianion. Thus, [Cr(III)(bpy(2-))(3)](3-) is a normal Werner-type Cr(III) (!) species. In all complexes containing (bpy(?))(1-) ligands, the ligand spins are strongly antiferromagnetically coupled to the spins of the central Cr(III) ion (d(3), S(Cr) = 3/2) affording the observed ground states given above. Thus, all redox chemistry of [Cr(bpy)(3)](n) complexes is ligand-based and documents that the ligand 2,2'-bipyridine is a redox noninnocent ligand; it exists in three oxidation levels in these complexes: as N,N'-coordinated neutral (bpy(0)), monoanionic π-radical (bpy(?))(1-), and diamagnetic dianionic (bpy(2-))(2-).     

Micropatterning topology on soft substrates affects myoblast proliferation and differentiation

Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 μm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 μm wide lanes compared to (39 ± 7)% in 500 μm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 μm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.     

Solvent‐ and Light‐Controlled Unidirectional Transit of a Nonsymmetric Molecular Axle Through a Nonsymmetric Molecular Wheel

The development of a pseudorotaxane motif capable of performing unidirectional threading and dethreading processes under control of external stimuli is particularly important for the construction of processive linear motors based on rotaxanes and, at least in principle, it discloses the possibility to access to rotary motors based on catenanes. Here, we report a strategy to obtain the solvent‐controlled unidirectional transit of a molecular axle through a molecular wheel. It is based on the use of appropriately designed molecular components, the essential feature of which is their non‐symmetric structure. Specifically they are an axle containing a central electron‐acceptor 4,4′‐bipyridinium core functionalized with a hexanol chain at one side, and a stilbene unit connected through a C6 chain at the other side, and a heteroditopic tris(phenylureido)‐calix[6]arene wheel. In apolar solvents the axle threads into the wheel from its upper rim and with the end carrying the OH group, giving an oriented pseudorotaxane structure. After a stoppering reaction, which replaces the small hydroxy group with a bulky diphenylacetyl moiety, and replacement of the apolar solvent with a polar one, dethreading occurs through the slippage of the stilbene unit from the lower rim of the wheel, that is, in the same direction of the threading process. The essential role played by the stilbene unit to achieve the unidirectional transit of the axle through the wheel, and to tune the dethreading rate by light is also demonstrated.