Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of β-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins’ Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.
Optical and X-ray diffraction characterizations of crystals I and II of (2Z)-2-(4-bromophenyl)-3-[4-(dimethylamino)phenyl]prop-2-enenitrile (Z-4-BrPhDMAPhACN) are reported. I and II belong to the same monoclinic space group and have nearly identical unit-cell dimensions [I: a = 11.0169(2); b = 6.02041(11); c = 21.8541(4); (β) 98.4082(18). II: a = 11.0475(6); b = 6.0273(3); c = 21.8533(11); (β) 98.315(5)]. Crystals I and II were formed under different conditions and have different crystal habits and colors: crystals I are yellow blocks, whereas crystals II are small, orange needles.I and II show absorption maxima (λ abs ) in solution at 404 nm and emission (λ em ) maxima at 501 nm (I) and at 502 nm (II). However, their emission maxima are different in the solid state: I shows an emission at 512 nm and a shoulder at 534 nm, whereas II exhibits a λ em maximum at 582 nm. The differences in solid-state photoluminescence were attributed to variations in the crystal morphology and to the crystal habit and size. Both I and II were characterized by nuclear magnetic resonance, mass spectrometry, infrared spectroscopy, UV−vis spectroscopy, fluorescence, cyclic voltammetry, single-crystal and powder X-ray diffraction, differential scanning calorimetry, scanning electron microscopy, and density functional theory calculations.
We report single crystal X-ray diffraction (hereafter, SCXRD) analyses of derivatives featuring the electron-donor N-ethylcarbazole or the (4-diphenylamino)phenyl moieties associated with a -CN group attached to a double bond. The compounds are (2Z)-3-(4-(diphenylamino)-phenyl)-2-(pyridin-3-yl)prop-2-enenitrile (I), (2Z)-3-(4-(diphenylamino)phenyl)-2-(pyridin-4-yl)-prop-2-enenitrile (II) and (2Z)-3-(9-ethyl-9H-carbazol-3-yl)-2-(pyridin-2-yl)enenitrile (III). SCXRD analyses reveal that I and III crystallize in the monoclinic space groups P2/c with Z' = 2 and C2/c with Z' = 1, respectively. Compound II crystallized in the orthorhombic space group Pbcn with Z' = 1. The molecular packing analysis was conducted to examine the pyridine core effect, depending on the ortho, meta- and para-positions of the nitrogen atom, with respect to the optical properties and number of independent molecules (Z'). It is found that the double bond bearing a diphenylamino moiety introduced properties to exhibit a strong π-π-interaction in the solid state. The compounds were examined to evaluate the effects of solvent polarity, the role of the molecular structure, and the molecular interactions on their self-assembly behaviors. Compound I crystallized with a cell with two conformers, anti and syn, due to interaction with solvent. DFT calculations indicated the anti and syn structures of I are energetically stable (less than 1 eV). Also electrochemical and photophysical properties of the compounds were investigated, as well as the determination of optimization calculations in gas and different solvent (chloroform, cyclohexane, methanol, ethanol, tetrahydrofuran, dichloromethane and dimethyl sulfoxide) in the Gaussian09 program. The effect of solvent by PCM method was also investigated. The frontier HOMO and LUMO energies and gap energies are reported.
γD-Crystallin (HγDC) is a key structural protein in the human lens, whose aggregation has been associated with the development of cataracts. Single-point mutations and post-translational modifications destabilize HγDC interactions, forming partially folded intermediates, where hydrophobic residues are exposed and thus triggering its aggregation. In this work, we used alchemical free-energy calculations to predict changes in thermodynamic stability (ΔΔG) of 10 alanine-scanning variants and 12 HγDC mutations associated with the development of congenital cataract. Our results show that W42R is the most destabilizing mutation in HγDC. This has been corroborated through experimental determination of ΔΔG employing differential scanning calorimetry. Calculations of hydration free energies from the HγDC WT and the W42R mutant suggested that the mutant has a higher aggregation propensity. Our combined theoretical and experimental results contribute to understand HγDC destabilization and aggregation mechanisms in age-onset cataracts.
We report an interesting and clear property-structure relationship between the emission wavelength and the morphologies of samples separated by evaporation using a sublimator and a solvent. The compounds are (Z)-3-(4-(dimethylamino)phenyl)-2-(pyridin-4-yl)acrylonitrile Z-DMPyACN (I) and (Z)-3-(4-(diphenylamino)phenyl)-2-(pyridin-2-yl)acrylonitrile Z-DPPyACN (II). I exhibits strong emission in the solid state, but not in a solution, whereas II exhibits emission in a solution and as a solid. Characterization by single-crystal X-ray crystallography showed that the Z-DMPyACN polymorph (Ii) has a monoclinic unit cell with a = 7.0445(2); b = 17.1474IJ5); c = 10.9776(4) Å, and β = 107.251IJ4)°and is found in the space group P2 1 /c with Z′ = 1. The orange crystal habit and color are different from the structure reported, (Z′ = 2, block and yellow). The Ii luminescence displayed an absorbance maximum at λ max = 427 nm and an emission maximum at λ em = 555 nm. From the Z-DPPyACN II evaporation in the sublimator, three solids were recovered, featuring emission wavelengths that were dependent on the size of the particles (nanoparticles) rather than on the single-crystal structure. The IIg, IIy, and IIo powders showed emission maxima at λ em = 546, 558, and 607 nm, respectively. Characterization was carried out by UV-vis, fluorescence and 1 H-NMR in different polarity solvents, as well as SCXRD, PXRD and SEM.
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