RESUMEN
Coumarin-based fluorescent agents play an important role in the manifold fundamental scientific and technological areas and need to be carefully studied. In this research, linear photophysics, photochemistry, fast vibronic relaxations, and two-photon absorption (2PA) of the coumarin derivatives, methyl 4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]butanoate (1) and methyl 4-[4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]phenoxy]butanoate (2), were comprehensively analyzed using stationary and time-resolved spectroscopic techniques, along with quantum-chemical calculations. The steady-state one-photon absorption, fluorescence emission, and excitation anisotropy spectra, as well as 3D fluorescence maps of 3-hetarylcoumarins 1 and 2 were obtained at room temperature in solvents of different polarities. The nature of relatively large Stokes shifts (â¼4000-6000 cm-1), specific solvatochromic behavior, weak electronic π â π* transitions, and adherence to Kasha's rule were revealed. The photochemical stability of 1 and 2 was explored quantitatively, and values of photodecomposition quantum yields, on the order of â¼10-4, were determined. A femtosecond transient absorption pump-probe technique was used for the investigation of fast vibronic relaxation and excited-state absorption processes in 1 and 2, while the possibility of efficient optical gain was shown for 1 in acetonitrile. The degenerate 2PA spectra of 1 and 2 were measured by an open aperture z-scan method, and the maximum 2PA cross-sections of â¼300 GM were obtained. The electronic nature of the hetaryl coumarins was analyzed by quantum-chemical calculations using DFT/TD-DFT level of theory and was found to be in good agreement with experimental data.
RESUMEN
d-aminoacyl-tRNA-deacylase (DTD) prevents the incorporation of d-amino acids into proteins during translation by hydrolyzing the ester bond between mistakenly attached amino acids and tRNAs. Despite extensive study of this proofreading enzyme, the precise catalytic mechanism remains unknown. Here, a combination of biochemical and computational investigations has enabled the discovery of a new substrate-assisted mechanism of d-Tyr-tRNATyr hydrolysis by Thermus thermophilus DTD. Several functional elements of the substrate, misacylated tRNA, participate in the catalysis. During the hydrolytic reaction, the 2'-OH group of the Ð76 residue of d-Tyr-tRNATyr forms a hydrogen bond with a carbonyl group of the tyrosine residue, stabilizing the transition-state intermediate. Two water molecules participate in this reaction, attacking and assisting ones, resulting in a significant decrease in the activation energy of the rate-limiting step. The amino group of the d-Tyr aminoacyl moiety is unprotonated and serves as a general base, abstracting the proton from the assisting water molecule and forming a more nucleophilic ester-attacking species. Quantum chemical methodology was used to investigate the mechanism of hydrolysis. The DFT-calculated deacylation reaction is in full agreement with the experimental data. The Gibbs activation energies for the first and second steps were 10.52 and 1.05â kcal/mol, respectively, highlighting that the first step of the hydrolysis process is the rate-limiting step. Several amino acid residues of the enzyme participate in the coordination of the substrate and water molecules. Thus, the present work provides new insights into the proofreading details of misacylated tRNAs and can be extended to other systems important for translation fidelity.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Biosíntesis de Proteínas/fisiología , ARN Bacteriano , Aminoacil-ARN de Transferencia , Thermus thermophilus , Proteínas Bacterianas/química , Catálisis , Hidrólisis , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Thermus thermophilus/química , Thermus thermophilus/metabolismoRESUMEN
Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNAPro. The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNAPro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , ARN de Transferencia/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Bacterias/enzimología , Bacterias/genética , Dominio Catalítico , Enlace de Hidrógeno , Hidrólisis , Modelos Moleculares , Conformación Molecular , Unión Proteica , ARN de Transferencia/metabolismo , Relación Estructura-ActividadRESUMEN
Self-assemblies formed by the new synthesized tricationic porphyrin derivative (TMPyP(3+)) on the polyanionic inorganic polyphosphate (PPS) in aqueous solution were studied using different spectroscopic techniques and DFT calculation method. From the fluorescence quenching of the bound TMPyP(3+) molecules and their Raman spectra we conclude that porphyrin chromophores form the stable π-π stacking-assemblies onto PPS polyanions. The transformation of the Soret band in absorption spectra at different PPS/TMPyP(3+)concentration ratios evidences that the assemblies are mixtures of J- and H-aggregates. Molecular modeling performed shows that the flexibility of PPS strand allows a realization of spiral or "face-to-face" one-dimensional structures formed by porphyrin molecules arranged in parallel and antiparallel modes. The peculiarity of PPS structure allows a formation of two porphyrin stacks on opposite sides of polymer strands that result in the appearance of higher-order aggregates. Their size was estimated from the light scattering data. Distinctions between TMPyP(3+) and TMPyP4 aggregation on PPS template are discussed.
Asunto(s)
Polifosfatos/química , Porfirinas/química , Fluorescencia , Luz , Modelos Moleculares , Polielectrolitos , Polímeros/química , Dispersión de RadiaciónRESUMEN
Ab initio quantum-chemical study of specific point contacts of replisome proteins with DNA modeled by acetic acid with canonical and mutagenic tautomers of DNA bases methylated at the glycosidic nitrogen atoms was performed in vacuo and continuum with a low dielectric constant (ϵ ⼠4) corresponding to a hydrophobic interface of protein-nucleic acid interaction. All tautomerized complexes were found to be dynamically unstable, because the electronic energies of their back-reaction barriers do not exceed zero-point vibrational energies associated with the vibrational modes whose harmonic vibrational frequencies become imaginary in the transition states of the tautomerization reaction. Additionally, based on the physicochemical arguments, it was demonstrated that the effects of biomolecular environment cannot ensure dynamic stabilization. This result allows suggesting that hypothetically generated by DNA-binding proteins of replisome rare tautomers will have no impact on the total spontaneous mutation due to the low reverse barrier allowing a quick return to the canonical form.