RESUMEN
Non-heme iron enzymes play key roles in antibiotic, neurotransmitter, and natural product biosynthesis, DNA repair, hypoxia regulation, and disease states. These enzymes had been refractory to traditional bioinorganic spectroscopic methods. Thus, we developed variable-temperature variable-field magnetic circular dichroism (VTVH MCD) spectroscopy to experimentally define the excited and ground ligand field states of non-heme ferrous enzymes (Solomon et al., 1995). This method provides detailed geometric and electronic structure insight and thus enables a molecular level understanding of catalytic mechanisms. Application of this method across the five classes of non-heme ferrous enzymes has defined that a general mechanistic strategy is utilized where O2 activation is controlled to occur only in the presence of all cosubstrates.
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Dominio Catalítico , Dicroismo Circular , Dicroismo Circular/métodos , Hierro/química , Hierro/metabolismo , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/metabolismo , Oxígeno/metabolismo , Oxígeno/química , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismoRESUMEN
The biotechnological potential of Rieske Oxygenases (ROs) and their cognate reductases remains unmet, in part because these systems can be functionally short-lived. Here, we describe a set of experiments aimed at identifying both the functional and structural stability limitations of ROs, using terephthalate (TPA) dioxygenase (from Comamonas strain E6) as a model system. Successful expression and purification of a cofactor-complete, histidine-tagged TPA dioxygenase and reductase protein system requires induction with the Escherichia coli host at stationary phase as well as a chaperone inducing cold-shock and supplementation with additional iron, sulfur, and flavin. The relative stability of the Rieske cluster and mononuclear iron center can then be assessed using spectroscopic and functional measurements following dialysis in an iron chelating buffer. These experiments involve measurements of the overall lifetime of the system via total turnover number using both UV-Visible absorbance and HPLC analyses, as well specific activity as a function of temperature. Important methods for assessing the stability of these multi-cofactor, multi-protein dependent systems at multiple levels of structure (secondary to quaternary) include differential scanning calorimetry, circular dichroism, and metallospectroscopy. Results can be rationalized in terms of three-dimensional structures and bioinformatics. The experiments described here provide a roadmap to a detailed characterization of the limitations of ROs. With a few notable exceptions, these issues are not widely addressed in current literature.
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Estabilidad de Enzimas , Oxigenasas/química , Oxigenasas/metabolismo , Oxigenasas/genética , Dicroismo Circular/métodos , Temperatura , Cromatografía Líquida de Alta Presión/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Espectrofotometría Ultravioleta/métodosRESUMEN
Controlling the assembly of high-order structures is central to soft-matter and biomaterial engineering. Angle-resolved linear dichroism can probe the ordering of chiral collagen molecules in the dense state. Collagen triple helices were aligned by solvent evaporation. Their ordering gives a strong linear dichroism (LD) that changes sign and intensity with varying sample orientations with respect to the beam linear polarization. Being complementary to circular dichroism, which probes the structure of chiral (bio)molecules, LD can shift from the molecular to the supramolecular scale and from the investigation of the conformation to interactions. Supported by multiphoton microscopy and X-ray scattering, we show that LD provides a straightforward route to probe collagen alignment, determine the packing density, and monitor denaturation. This approach could be adapted to any assembly of chiral (bio)macromolecules, with key advantages in detecting large-scale assemblies with high specificity to aligned and chiral molecules and improved sensitivity compared to conventional techniques.
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Materiales Biocompatibles , Dicroismo Circular , Colágeno , Materiales Biocompatibles/química , Colágeno/química , Dicroismo Circular/métodos , Animales , Difracción de Rayos X/métodosRESUMEN
Circular dichroism (CD) is a spectroscopic technique commonly used for the analysis of proteins. Particularly, it allows the determination of protein secondary structure content in various media, including the membrane environment. In this chapter, we present how CD applications can be used to analyze the interaction of proteins with bacterial outer membrane vesicles (OMVs). Most CD studies characterizing the structure of proteins inserted into membranes rely on artificial lipid bilayers, mimicking natural membranes. Nevertheless, these artificial models lack the important features of the true membrane, especially for the outer membrane of Gram-negative bacteria. These features include lipid diversity, glycosylation, and asymmetry. Here, we show how to analyze the interactions of proteins, either integral or peripheral, with OMVs in solution and with supported membranes of OMVs, using conventional CD and orientated circular dichroism (OCD). We explain how to decipher the spectroscopic signals to obtain information on the molecular structure of the protein upon its interaction with an OMV and through its potential insertion into an OMV membrane.
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Proteínas de la Membrana Bacteriana Externa , Dicroismo Circular , Sincrotrones , Dicroismo Circular/métodos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Estructura Secundaria de Proteína , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/químicaRESUMEN
Prokaryotes organize intracellular compartments with protein-based organelles called encapsulins. Encapsulins with icosahedral symmetry can encapsulate specific cargo proteins mediated by targeting peptides or encapsulation-mediating domains. Encapsulins have been used in eukaryotic cells for bioengineering, vaccine development, and nanoparticle alignment. Their versatility makes them attractive for research; however, detailed structural information on encapsulins is crucial for further applied research. However, cargo proteins are randomly oriented inside the icosahedral encapsulins. The random orientation of cargo proteins presents a challenge for structural analysis that relies on averaging processes such as x-ray crystallography and cryo-electron microscopy (cryo-EM) single-particle imaging. Therefore, we aimed to accurately estimate the secondary structure content and elucidate the structure of cargo proteins inside the particle by measuring the circular dichroism (CD) spectra using vacuum ultraviolet circular dichroism (VUVCD) spectroscopy. Thus, the structure of the cargo protein inside encapsulin was evaluated. This approach could potentially set a standard for evaluating cargo proteins inside particles in future applied research on encapsulins.
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Dicroismo Circular , Dicroismo Circular/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Lobeline (LOB), a naturally occurring alkaloid, has a broad spectrum of pharmacological activities and therapeutic potential, including applications in central nervous system disorders, drug misuse, multidrug resistance, smoking cessation, depression, and epilepsy. LOB represents a promising compound for developing treatments in various medical fields. However, despite extensive pharmacological profiling, the biophysical interaction between the LOB and proteins remains largely unexplored. In the current article, a range of complementary photophysical and cheminformatics methodologies were applied to study the interaction mechanism between LOB and the carrier protein HSA. Steady-state fluorescence and fluorescence lifetime experiments confirmed the static-quenching mechanisms in the HSA-LOB system. "K" (binding constant) of the HSA-LOB system was determined to be 105 M-1, with a single preferable binding site in HSA. The forces governing the HSA-LOB stable complex were analyzed by thermodynamic parameters and electrostatic contribution. The research also investigated how various metal ions affect complex binding. Site-specific binding studies depict Site I as probable binding in HSA by LOB. We conducted synchronous fluorescence, 3D fluorescence, and circular dichroism studies to explore the structural alteration occurring in the microenvironment of amino acids. To understand the robustness of the HSA-LOB complex, we used theoretical approaches, including molecular docking and MD simulations, and analyzed the principal component analysis and free energy landscape. These comprehensive studies of the structural features of biomolecules in ligand binding are of paramount importance for designing targeted drugs and delivery systems.
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Dicroismo Circular , Interacciones Hidrofóbicas e Hidrofílicas , Lobelina , Unión Proteica , Albúmina Sérica Humana , Termodinámica , Humanos , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo , Lobelina/química , Lobelina/metabolismo , Sitios de Unión , Dicroismo Circular/métodos , Conformación Proteica , Espectrometría de Fluorescencia , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/químicaRESUMEN
Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation platform of oligonucleotides for acquiring a comprehensive view of their properties have been limited. As the chemical stability and the efficacy as well as the solution properties at a high concentration should be related to their higher-order structure and intra-/intermolecular interactions, their detailed understanding enables effective formulation development. Here, the higher-order structure and the thermodynamic stability of the thrombin-binding aptamer (TBA) and four modified TBAs, which have similar sequences but were expected to have different higher-order structures, were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). Then, the relationship between the higher-order structure and the solution properties including solubility, viscosity, and stability was investigated. The impact of the higher-order structure on the antithrombin activity was also confirmed. The higher-order structure and intra-/intermolecular interactions of the oligonucleotides were affected by types of buffers because of different potassium concentrations, which are crucial for the formation of the G-quadruplex structure. Consequently, solution properties, such as solubility and viscosity, chemical stability, and antithrombin activity, were also influenced. Each instrumental analysis had a complemental role in investigating the higher-order structure of TBA and modified TBAs. The utility of each physicochemical characterization method during the preclinical developmental stages is also discussed.
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Aptámeros de Nucleótidos , Dicroismo Circular , Oligonucleótidos , Aptámeros de Nucleótidos/química , Dicroismo Circular/métodos , Oligonucleótidos/química , Rastreo Diferencial de Calorimetría/métodos , Viscosidad , Espectroscopía de Resonancia Magnética/métodos , Solubilidad , Termodinámica , G-Cuádruplex , Estabilidad de Medicamentos , HumanosRESUMEN
A simplified molecular-dynamics-based electronic circular dichroism (ECD) approach was tested on three condensed derivatives with limited conformational flexibility and an isochroman-2H-chromene hybrid, the ECD spectra of which could not be precisely reproduced by the conventional ECD calculation protocol. Application of explicit solvent molecules at the molecular mechanics (MD) level in the dynamics simulations and subsequent TDDFT-ECD calculation for the unoptimized MD structures was able to improve the agreements between experimental and computed spectra. Since enhancements were achieved even for molecules with limited conformational flexibility, deformations caused by the solvent molecules and multitudes of conformers produced with unoptimized geometries seem to be key factors for better agreement. The MD approach could confirm that aggregation of the phenanthrene natural product luzulin A had a significant contribution to a specific wavelength range of the experimental ECD. The MD approach has proved that dimer formation occurred in solution and this was responsible for the anomalous ECD spectrum. The scope and limitations of the method have also been discussed.
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Dicroismo Circular , Simulación de Dinámica Molecular , Dicroismo Circular/métodos , Fenantrenos/química , Conformación Molecular , Solventes/químicaRESUMEN
Hemp-sprouts are emerging as a new class of attractive functional food due to their numerous health benefits when compared to other sprout species. Indeed, the high content of beneficial components including polyphenols and flavonoids makes this type of food a promising and successful market. However, the available literature on this topic is limited and often conflicting as regards to the content of phytocannabinoids. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was applied in an untargeted metabolomics fashion to extracts of hemp seeds, sprouts and microgreens of nine different genotypes. Both unsupervised and supervised multivariate statistical analysis was performed to reveal variety-specific profiles of phytocannabinoids with surprisingly remarkable levels of phytocannabinoids even in chemotype V samples. Furthermore, a targeted HPLC-HRMS analysis was carried out for the quantitative determination of the major phytocannabinoids including CBDA, CBD, CBGA, CBG, CBCA, CBC, THCA, and trans-Δ9-THC. The last part of the study was focused on the evaluation of the enantiomeric composition of CBCA in hemp seeds, sprouts and microgreens in the different varieties by HPLC-CD (HPLC with online circular dichroism). Chiral analysis of CBCA showed a wide variability of its enantiomeric composition in the different varieties, thus contributing to the understanding of the intriguing stereochemical behavior of this compound in an early growth stage. However, further investigation is needed to determine the genetic factors responsible for the low enantiopurity of this compound.
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Cannabis , Semillas , Cannabis/química , Cannabis/crecimiento & desarrollo , Semillas/química , Cromatografía Líquida de Alta Presión/métodos , Cannabinoides/análisis , Cannabinoides/química , Extractos Vegetales/química , Extractos Vegetales/análisis , Espectrometría de Masas/métodos , Metabolómica/métodos , Estereoisomerismo , Dicroismo Circular/métodosRESUMEN
Metal clusters have drawn considerable research attention over the years due to their fascinating optical properties. Owing to their appealing photophysical characteristics, these materials have drawn attention as potential candidates for various application in diverse fields, including disease detection, biosensing, chemical sensing, and the fabrication of light-harvesting materials. Presently, there is an increasing research focus on the use of clusters in biomedical research, both as biodetection platform and as bioimaging agents. Of special interest are chiral clusters, which can selectively interact with chiral biomolecules owing to their optical activity. Herein, we showcase the use of a pair of chiroptically active copper clusters for the enantioselective detection of lysine, an amino acid of vast biological relevance. Two techniques are concurrently employed for the detection of lysine at varying concentrations. Circular dichroism serves as a potent tool for detecting lysine at low concentrations, whereas luminescence is effectively employed as a detection method for high analyte concentrations. The combined electronic impact of clusters and lysine resulted in the emergence of an enhanced enantioselective Cotton effect at specific wavelength.
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Cobre , Lisina , Lisina/química , Lisina/análisis , Cobre/química , Cobre/análisis , Estereoisomerismo , Dicroismo Circular/métodosRESUMEN
Fluorescence spectroscopy serves as a vital technique for studying the interaction between light and fluorescent molecules. It encompasses a range of methods, each presenting unique advantages and applications. This technique finds utility in various chemical studies. This review discusses Fluorescence spectroscopy, its branches such as Time-Resolved Fluorescence Spectroscopy (TRFS) and Fluorescence Lifetime Imaging Microscopy (FLIM), and their integration with other spectroscopic methods, including Raman, Infrared (IR), and Circular Dichroism (CD) spectroscopies. By delving into these methods, we aim to provide a comprehensive understanding of the capabilities and significance of fluorescence spectroscopy in scientific research, highlighting its diverse applications and the enhanced understanding it brings when combined with other spectroscopic methods. This review looks at each technique's unique features and applications. It discusses the prospects of their combined use in advancing scientific understanding and applications across various domains.
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Espectrometría de Fluorescencia , Espectrometría Raman , Espectrometría Raman/métodos , Espectrometría de Fluorescencia/métodos , Dicroismo Circular/métodos , Espectrofotometría Infrarroja/métodos , HumanosRESUMEN
This study describes the interaction of human serum albumin (HSA) with the binol derivative (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (R-BrB), which has its optical activity based on the prohibitive energetic barrier for conversion into the enantiomer (S)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (S-BrB). The objective was to assess the ability of HSA to differentiate axial enantiomers based on their binding efficiency and their impact on the CD spectra. We discovered that both enantiomers were effective ligands, and the CD signal disappeared when equimolar amounts of R-BrB and S-BrB were simultaneously added, indicating no preference for either enantiomer. The complexation resulted in a significant signal increase at 250 nm and a bathochromic effect at 370 nm. Molecular docking simulations were performed, and the lower energy pose of R-BrB was selected for DFT calculations. The theoretical CD spectra of free and complexed R-BrB were obtained and showed alterations corroborating the experimental results. By comparing the difference spectrum (HSA:R-BrB minus HSA) with the spectrum of free RBrB in water or ethyl alcohol, we concluded that the CD signal intensification was due to the increased solubilization of R-BrB upon binding to HSA.
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Dicroismo Circular , Simulación del Acoplamiento Molecular , Naftoles , Albúmina Sérica Humana , Dicroismo Circular/métodos , Naftoles/química , Albúmina Sérica Humana/química , Estereoisomerismo , Humanos , Teoría Funcional de la Densidad , Simulación por Computador , Unión ProteicaRESUMEN
The misuse of pharmaceuticals has significantly increased in recent decades, becoming a major public health concern. The risks associated with medication misuse are particularly high in cases of overdose, especially when the active substances are chiral, as enantioselectivity plays an important role in toxicity. Promethazine (PMZ) is a chiral antihistamine marketed as a racemate and it is misused in "Purple Drank", a recreational drug beverage, that combines codeine and/or PMZ, with soda or alcohol leading to serious health consequences and fatalities in consumers around the world, particularly among teenagers. Information regarding the enantioselectivity in the toxicity of (R,S)-PMZ and its main metabolites, namely promethazine sulfoxide (PMZSO) and desmonomethyl promethazine (DMPMZ), is unknown. This work reported, for the first time, the enantioseparation, in milligram scale, of (R,S)-PMZ, (R,S)-DMPMZ, (R,S)- PMZSO and the determination of their absolute configurations by electronic circular dichroism (ECD). The enantioseparation of all the six enantiomers was accomplished in a homemade semi-preparative column with amylose tris-3,5-dimethylphenylcarbamate (AD) coated with aminopropyl Nucleosil silica. The enantiomeric purity was evaluated using the analytical Lux® 3⯵m i-Amylose-3 column, yielding enantiomeric purity values ranging between 94.4% and 99.7%. The elution order of all the enantiomers was accomplished combining the ECD results with an optical rotation detector. The elution order of the enantiomers was influenced only by the chiral selector, rather than the mobile phase. The cytotoxicity of the racemates and the isolated enantiomers towards differentiated SH-SY5Y cells was evaluated. (R,S)-DMPMZ exhibited a significantly higher cytotoxicity than (R,S)-PMZ, suggesting the metabolic bioactivation of (R,S)-PMZ. Conversely, no significant cytotoxicity was found for (R,S)-PMZSO, underscoring a metabolic detoxification pathway. Remarkably, enantioselectivity was observed for the cytotoxicity of PMZ; (R)-PMZ was significantly more cytotoxic than (S)-PMZ. The results underscore the importance to isolate the enantiomers in their enantiomerically form and their correct identification for toxicity enantioselectivity studies, which are vital to understand the drug's behaviour and safety, especially in case of overdoses.
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Prometazina , Prometazina/química , Estereoisomerismo , Humanos , Línea Celular Tumoral , Dicroismo Circular/métodos , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión/métodosRESUMEN
The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.
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Diseño de Fármacos , Péptidos , Péptidos/química , Dicroismo Circular/métodos , Estabilidad de Medicamentos , Secuencia de Aminoácidos , Cinética , Ácidos Aminoisobutíricos/química , Estabilidad Proteica , Espectrometría de Masas/métodosRESUMEN
The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.
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ADN/química , G-Cuádruplex , Guanina/química , Riboflavina/química , Dicroismo Circular/métodos , Guanina/análogos & derivados , Humanos , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica Molecular , Oxidación-Reducción , Regiones Promotoras Genéticas , Telómero/químicaRESUMEN
The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 104 M-1 has been assessed through a Stern-Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.
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MicroARNs/química , MicroARNs/genética , Albúmina Sérica Humana/química , Sitios de Unión/efectos de los fármacos , Dicroismo Circular/métodos , Biología Computacional/métodos , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Simulación del Acoplamiento Molecular/métodos , Simulación de Dinámica Molecular , Unión Proteica/fisiología , Albúmina Sérica Humana/metabolismo , Albúmina Sérica Humana/ultraestructura , Espectrometría de Fluorescencia/métodos , TermodinámicaRESUMEN
Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Disulfuros/metabolismo , Amiloide/química , Péptidos beta-Amiloides/química , Dicroismo Circular/métodos , Disulfuros/química , Humanos , Enlace de Hidrógeno , Microscopía Electrónica de Transmisión/métodos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Conformación Proteica en Lámina betaRESUMEN
Queuosine (Q) is a highly modified nucleoside of transfer RNA that is formed from guanosine triphosphate over the course of eight steps. The final step in this process, involving the conversion of epoxyqueuosine (oQ) to Q, is catalyzed by the enzyme QueG. A recent X-ray crystallographic study revealed that QueG possesses the same cofactors as reductive dehalogenases, including a base-off Co(II)cobalamin (Co(II)Cbl) species and two [4Fe-4S] clusters. While the initial step in the catalytic cycle of QueG likely involves the formation of a reduced Co(I)Cbl species, the mechanisms employed by this enzyme to accomplish the thermodynamically challenging reduction of base-off Co(II)Cbl to Co(I)Cbl and to convert oQ to Q remain unknown. In this study, we have used electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies in conjunction with whole-protein quantum mechanics/molecular mechanics (QM/MM) computations to further characterize wild-type QueG and select variants. Our data indicate that the Co(II)Cbl cofactor remains five-coordinate upon substrate binding to QueG. Notably, during a QM/MM optimization of a putative QueG reaction intermediate featuring an alkyl-Co(III) species, the distance between the Co ion and coordinating C atom of oQ increased to >3.3 Å and the C-O bond of the epoxide reformed to regenerate the oQ-bound Co(I)Cbl reactant state of QueG. Thus, our computations indicate that the QueG mechanism likely involves single-electron transfer from the transient Co(I)Cbl species to oQ rather than direct Co-C bond formation, similar to the mechanism that has recently been proposed for the tetrachloroethylene reductive dehalogenase PceA.
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Nucleósido Q/análogos & derivados , Oxidorreductasas/química , Bacillus subtilis , Catálisis , Dicroismo Circular/métodos , Cobalto/química , Cristalografía por Rayos X/métodos , Teoría Funcional de la Densidad , Espectroscopía de Resonancia por Spin del Electrón/métodos , Modelos Moleculares , Nucleósido Q/química , ARN de Transferencia/química , Vitamina B 12/químicaRESUMEN
Life on Earth employs chiral amino acids in stereochemical L-form, but the cause of molecular symmetry breaking remains unknown. Chiroptical properties of amino acids - expressed in circular dichroism (CD) - have been previously investigated in solid and solution phase. However, both environments distort the intrinsic charge distribution associated with CD transitions. Here we report on CD and anisotropy spectra of amino acids recorded in the gas phase, where any asymmetry is solely determined by the genuine electromagnetic transition moments. Using a pressure- and temperature-controlled gas cell coupled to a synchrotron radiation CD spectropolarimeter, we found CD active transitions and anisotropies in the 130-280 nm range, which are rationalized by ab initio calculation. As gas phase glycine was found in a cometary coma, our data may provide insights into gas phase asymmetric photochemical reactions in the life cycle of interstellar gas and dust, at the origin of the enantiomeric selection of life's L-amino acids.
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Aminoácidos/química , Dicroismo Circular/métodos , Gases/química , Anisotropía , Química Computacional , Glicina , Origen de la Vida , Fotoquímica , Estereoisomerismo , SincrotronesRESUMEN
All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions (CTR). Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region (IDPR). This curiosity has led us to investigate the cytosolic CTR of the spike glycoprotein of SARS-CoV-2 in isolation. The spike CTR is supposed to be from 1235 to 1273 residues or 1242-1273 residues based on our used prediction. Therefore, we have demonstrated the structural conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm. We believe that our findings will surely help in understanding the structure-function relationship of the spike protein's cytosolic region.