Structure-based dissection of the active site chemistry of leukotriene A4 hydrolase: implications for M1 aminopeptidases and inhibitor design

M1 aminopeptidases comprise a large family of biologically important zinc enzymes. We show that peptide turnover by the M1 prototype, leukotriene A4 hydrolase/aminopeptidase, involves a shift in substrate position associated with exchange of zinc coordinating groups, while maintaining the overall coordination geometry. The transition state is stabilized by residues conserved among M1 members and in the final reaction step, Glu-296 of the canonical zinc binding HEXXH motif shuffles a proton from the hydrolytic water to the leaving group. Tripeptide substrates bind along the conserved GXMEN motif, precisely occupying the distance between Glu-271 and Arg-563, whereas the Arg specificity is governed by a narrow S1 pocket capped with Asp-375. Our data provide detailed insights to the active site chemistry of M1 aminopeptidases and will aid in the development of novel enzyme inhibitors.     

The sugar conformation governs (6-4) photoproduct formation at the dinucleotide level

The LNA dinucleotide mimic of TpT whose two-sugar puckers are locked in the C3'-endo conformation selectively produces the corresponding cyclobutane pyrimidine dimer under 254 nm irradiation. In the natural series (TpT) the sugar puckers are in a major C2'-endo sugar conformation and the (6-4) photoproduct is also produced. Consequently, this study demonstrates that the C2'-endo conformation of the sugar pucker is necessary for (6-4) photoproduct formation.     

Dissolution and storage stability of nanostructured calcium carbonates and phosphates for nutrition

Rapid calcium (Ca) dissolution from nanostructured Ca phosphate and carbonate (CaCO₃) powders may allow them to be absorbed in much higher fraction in humans. Nanosized Ca phosphate and CaCO₃ made by flame-assisted spray pyrolysis were characterized by nitrogen adsorption, X-ray diffraction (XRD), Raman spectroscopy, and transmission electron microscopy. As-prepared nanopowders contained both CaCO₃ and CaO, but storing them under ambient conditions over 130 days resulted in a complete transformation into CaCO₃, with an increase in both crystal and particle sizes. The small particle size could be stabilized against such aging by cation (Mg, Zn, Sr) and anion (P) doping, with P and Mg being most effective. Calcium phosphate nanopowders made at Ca:P ≤ 1.5 were XRD amorphous and contained γ-Ca₂P₂O₇ with increasing hydroxyapatite content at higher Ca:P. Aging of powders with Ca:P = 1.0 and 1.5 for over 500 days gradually increased particle size (but less than for CaCO₃) without a change in phase composition or crystallinity. In 0.01 M H₃PO₄ calcium phosphate nanopowders dissolved ≈4 times more Ca than micronsized compounds and about twice more Ca than CaCO₃ nanopowders, confirming that nanosizing and/or amorphous structuring sharply increases Ca powder dissolution. Because higher Ca solubility in vitro generally leads to greater absorption in vivo, these novel FASP-made Ca nanostructured compounds may prove useful for nutrition applications, including supplementation and/or food fortification.     

Synthesis of functionalized de novo designed 8-16 kDa model proteins towards metal ion-binding and esterase activity

De novo design and total chemical synthesis of proteins provides a powerful approach for biological and biophysical studies with the ability to prepare artificial proteins with tailored properties, potentially of importance for biophysical studies, material science, nanobioscience, and as molecular probes. In this paper, the previously developed concept of carbohydrates as templates is employed in the de novo design of model proteins (artificial helix bundles) termed 'carboproteins'. The 4-alpha-helix bundle is a macromolecular structure, where four amphiphilic alpha-helical peptide strands form a hydrophobic core. Here this structure is modified towards achieving metal ion-binding and catalytic activity. We report: (i) test of directional effects from different tetravalent carbohydrate templates, (ii) synthesis and evaluation of carboproteins functionalized with phenol, pyridyl or imidazolyl moieties as potential ligands for metal ion-binding as well as for catalysis. Our results include: (i) support of our previous 'controversial' finding that for some carboproteins the degree of alpha-helicity depends on the template, i.e., that there is, to some extent, a controlling effect from the template, (ii) demonstration of binding of Cu(ii) to tetra-functional carboproteins by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), UV-VIS absorption spectroscopy and size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS); (iii) a kinetic investigation of the esterase activity.     

Modeling force-induced bio-polymer unfolding

We study the conformations of polymer chains in a poor solvent, with and without bending rigidity, by means of a simple statistical mechanics model. This model can be exactly solved for chains of length up to N = 55 using exact enumeration techniques. We analyze in detail the differences between the constant force and constant distance ensembles for large but finite N. At low temperatures, and in the constant force ensemble, the force–extension curve shows multiple plateaus (intermediate states), in contrast with the abrupt transition to an extended state prevailing in the N → ∞ limit. In the constant distance ensemble, the same curve provides a unified response to pulling and compressing forces, and agrees qualitatively with recent experimental results. We identify a cross-over length, proportional to N, below which the critical force of unfolding decreases with temperature, while above, it increases with temperature. Finally, the force–extension curve for stiff chains exhibits “saw-tooth” like behavior, as observed in protein unfolding experiments.     

Synthesis of substituted 2-cyanoarylboronic esters

The synthesis of substituted 2-cyanoarylboronic esters is described via lithiation/in situ trapping of the corresponding methoxy-, trifluoromethyl-, fluoro-, chloro-, and bromobenzonitriles. The crude arylboronic esters were obtained in high yields and purities and with good regioselectivities.     

Tuning quadratic nonlinear photonic crystal fibers for zero group-velocity mismatch

We consider an index-guiding silica photonic crystal fiber with a triangular hole pattern and a periodically poled quadratic nonlinearity. By tuning the pitch and the relative hole size, second-harmonic generation with zero group-velocity mismatch is found for any fundamental wavelength above 780 nm. The nonlinear strength is optimized when the fundamental has maximum confinement in the core. The conversion bandwidth allows for femtosecond-pulse conversion, and 4%-180%W(-1)cm(-2) relative efficiencies were found.