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Dry reforming of methane (DRM) has recently received wide attention owing to its outstanding performance in the reduction and conversion of CH4 and CO2 to syngas (H2 and CO). From an industrial perspective, nickel (Ni)-supported catalysts have been deemed among the most suitable catalysts for DRM owing to their low cost and high activity compared to noble metals. However, a downside of nickel catalysts is their high susceptibility to deactivation due to coke formation and sintering at high temperatures. Using appropriate supports and preparation methods plays a major role in improving the activity and stability of Ni-supported catalysts. Halloysite nanotubes (HNTs) are largely utilized in catalysis as a support for Ni owing to their abundance, low cost, and ease of preparation. The treatment of HNTs (chemical or physical) prior to doping with Ni is considered a suitable method for increasing the overall performance of the catalyst. In this study, the surface of HNTs was activated with acids (HNO3 and H2SO4) and alkalis (NaOH and Na2CO3 + NaNO3) prior to Ni doping to assess the effects of support treatment on the stability, activity, and longevity of the catalyst. Nickel catalysts on raw HNT, acid-treated HNT, and alkali-treated HNT supports were prepared via wet impregnation. A detailed characterization of the catalysts was conducted using X-ray diffraction (XRD), BET surface area analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), solid-state nuclear magnetic resonance (ssNMR), H2-temperature programmed reduction, (H2-TPR), CO2-temperature programmed desorption (CO2-TPD), and Ni-dispersion via H2-pulse chemisorption. Our results reveal a clear alteration in the structure of HNTs after treatment, while elemental mapping shows a uniform distribution of Ni throughout all the different supports. Moreover, the supports treated with a molten salt method resulted in the overall highest CO2 and CH4 conversion among the studied catalysts and exhibited high stability over 24 hours testing.
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Non-alcoholic fatty liver disease (NAFLD) is a common liver lesion that is untreatable with medications. Glucagon-like peptide-1 receptor (GLP-1R) agonists have recently emerged as a potential NAFLD pharmacotherapy. However, the molecular mechanisms underlying these drugs' beneficial effects are not fully understood. Using Fourier transform infrared (FTIR) spectroscopy, we sought to investigate the biochemical changes in a steatosis cell model treated or not with the GLP-1R agonist Exendin-4 (Ex-4). HepG2 cells were made steatotic with 400 µM of oleic acid and then treated with 200 nM Ex-4 in order to reduce lipid accumulation. We quantified steatosis using the Oil Red O staining method. We investigated the biochemical alterations induced by steatosis and Ex-4 treatment using Fourier transform infrared (FTIR) spectroscopy and chemometric analyses. Analysis of the Oil Red O staining showed that Ex-4 significantly reduces steatosis. This reduction was confirmed by FTIR analysis, as the phospholipid band (C=O) at 1740 cm-1 in Ex-4 treated cells is significantly decreased compared to steatotic cells. The principal component analysis score plots for both the lipid and protein regions showed that the untreated and Ex-4-treated samples, while still separated, are clustered close to each other, far from the steatotic cells. The biochemical and structural changes induced by OA-induced lipotoxicity are at least partially reversed upon Ex-4 treatment. FTIR and chemometric analyses revealed that Ex-4 significantly reduces OA-induced lipid accumulation, and Ex-4 also restored the lipid and protein biochemical alterations caused by lipotoxicity-induced oxidative stress. In combination with chemometric analyses, FTIR spectroscopy may offer new approaches for investigating the mechanisms underpinning NAFLD.
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Interactions between programmed death-1 (PD-1) with its ligand PD-L1 on tumor cells can antagonize T cell responses. Inhibiting these interactions using immune checkpoint inhibitors has shown promise in cancer immunotherapy. MDA-MB-231 is a triple negative breast cancer cell line that expresses PD-L1. In this study, we investigated the biochemical changes in MDA-MB-231 cells following treatment with atezolizumab, a specific PD-L1 blocker. Our readouts were Fourier Transform Infrared (FTIR) spectroscopy and flow cytometric analyses. Chemometrical analysis, such as principal component analysis (PCA), was applied to delineate the spectral differences. We were able to identify the chemical alterations in both protein and lipid structure of the treated cells. We found that there was a shift from random coil and α-helical structure to ß-sheet conformation of PD-L1 on tumor cells due to atezolizumab treatment, which could hinder binding with its receptors on immune cells, ensuring sustained T cell activation for potent immune responses. This work provides novel information about the effects of atezolizumab at molecular and cellular levels. FTIR bio-spectroscopy, in combination with chemometric analyses, may expedite research and offer new approaches for cancer immunology.
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Two-dimensional (2D) tungsten disulfide (WS2) quantum dots offer numerous promising applications in materials and optoelectronic sciences. Additionally, the catalytic and photoluminescence properties of ultra-small WS2 nanoparticles are of potential interest in biomedical sciences. Addressing the use of WS2 in the context of infection, the present study describes the conjugation of two potent antimicrobial peptides with WS2 quantum dots, as well as the application of the resulting conjugates in antimicrobial therapy and bioimaging. In doing so, we determined the three-dimensional solution structure of the quantum dot-conjugated antimicrobial peptide by a series of high-resolution nuclear magnetic resonance (NMR) techniques, correlating this to the disruption of both model lipid and bacterial membranes, and to several key biological performances, including antimicrobial and anti-biofilm effects, as well as cell toxicity. The results demonstrate that particle conjugation enhances the antimicrobial and anti-biofilm potency of these peptides, effects inferred to be due to multi-dendate interactions for the conjugated peptides. As such, our study provides information on the mode-of-action of such conjugates, laying the foundation for their potential use in treatment and monitoring of infections.
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Antiinfecciosos/farmacología , Diagnóstico por Imagen , Disulfuros/química , Péptidos/química , Puntos Cuánticos/química , Tungsteno/química , Secuencia de Aminoácidos , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/ultraestructura , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/ultraestructuraRESUMEN
Cationic antimicrobial peptides (AMPs) are emerging as effective alternatives to conventional therapeutics that are used against the ever-rising number of multidrug-resistant microbial strains. Most studies established the peptide's amphipathicity and electrostatic interaction with the membrane as the basis for their antimicrobial effect. However, the interplay between the stoichiometric ratio of lipids, local geometry, diverse physicochemical properties of the host membranes and antimicrobial peptide efficacy is still poorly understood. In the present study, we investigate the mechanism of interaction of VG16KRKP (VARGWKRKCPLFGKGG), a novel AMP designed from the dengue-virus fusion peptide, with bacterial/fungal membrane mimics. Fluorescence based dye leakage assays show that membrane disruption is not solely induced by electrostatic interaction but also driven by stoichiometric ratio of the lipids that dictates the net surface charge, amount of lipid defects and local geometry of the membrane. Solid-state 14N and 31P NMR experiments show that peptide interaction results in lowering of lipid order around both the headgroups and acyl chains, suggesting deep peptide insertion. Further, an increase or decrease in membrane stability of the host membrane was observed in differential scanning calorimetry (DSC) thermograms, dictated by the overall stoichiometric ratio of the lipids and the sterol present. In general, our results help understand the diverse fates of host membranes against an antimicrobial peptide.
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Péptidos Catiónicos Antimicrobianos/química , Virus del Dengue/química , Membranas Artificiales , Proteínas Virales/químicaRESUMEN
Atomically resolved crystal structures not only suffer from the inherent uncertainty in accurately locating H atoms but also are incapable of fully revealing the underlying forces enabling the formation of final structures. Therefore, the development and application of novel techniques to illuminate intermolecular forces in crystalline solids are highly relevant to understand the role of hydrogen atoms in structure adoption. Novel developments in 1H NMR MAS methodology can now achieve robust measurements of 1H chemical shift anisotropy (CSA) tensors which are highly sensitive to electrostatics. Herein, we use 1H CSA tensors, measured by MAS experiments and characterized using DFT calculations, to reveal the structure-driving factors between the two polymorphic forms of acetaminophen (aka Tylenol or paracetamol) including differences in hydrogen bonding and the role of aromatic interactions. We demonstrate how the 1H CSAs can provide additional insights into the static picture provided by diffraction to elucidate rigid molecules.
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Multidrug resistance against the existing antibiotics is one of the most challenging threats across the globe. Antimicrobial peptides (AMPs), in this regard, are considered to be one of the effective alternatives that can overcome bacterial resistance. MSI-594, a 24-residue linear alpha-helical cationic AMP, has been shown to function via the carpet mechanism to disrupt bacterial membrane systems. To better understand the role of lipid composition in the function of MSI-594, in the present study, eight different model membrane systems have been studied using accelerated molecular dynamics (aMD) simulations. The simulated results are helpful in discriminating the particular effects of cationic MSI-594 against zwitterionic POPC, anionic POPG and POPS, and neutral POPE lipid moieties. Additionally, the effects of various heterogeneous POPC/POPG (7 : 3), POPC/POPS (7 : 3), and POPG/POPE (1 : 3 and 3 : 1) bilayer systems on the dynamic interaction of MSI-594 have also been investigated. The effect on the lipid bilayer due to the interaction with the peptide is characterized by lipid acyl-chain order, membrane thickness, and acyl-chain dynamics. Our simulation results show that the lipid composition affects the membrane interaction of MSI-594, suggesting that membrane selectivity is crucial to its mechanism of action. The results reported in this study are helpful to obtain accurate atomistic-level information governing MSI-594 and its membrane disruptive antimicrobial mechanism of action, and to design next generation potent antimicrobial peptides.
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Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular , Péptidos/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Péptidos/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Fosfatidilserinas/química , Estructura Secundaria de ProteínaRESUMEN
Protons are vastly abundant in a wide range of exciting macromolecules and thus can be a powerful probe to investigate the structure and dynamics at atomic resolution using solid-state NMR (ssNMR) spectroscopy. Unfortunately, the high signal sensitivity, afforded by the high natural-abundance and high gyromagnetic ratio of protons, is greatly compromised by severe line broadening due to the very strong 1H-1H dipolar couplings. As a result, protons are rarely used, in spite of the desperate need for enhancing the sensitivity of ssNMR to study a variety of systems that are not amenable for high resolution investigation using other techniques including X-ray crystallography, cryo-electron microscopy, and solution NMR spectroscopy. Thanks to the remarkable improvement in proton spectral resolution afforded by the significant advances in magic-angle-spinning (MAS) probe technology, 1H ssNMR spectroscopy has recently attracted considerable attention in the structural and dynamics studies of various molecular systems. However, it still remains a challenge to obtain narrow 1H spectral lines, especially from proteins, without resorting to deuteration. In this Account, we review recent proton-based ssNMR strategies that have been developed in our laboratory to further improve proton spectral resolution without resorting to chemical deuteration for the purposes of gaining atomistic-level insights into molecular structures of various crystalline solid systems, using small molecules and peptides as illustrative examples. The proton spectral resolution enhancement afforded by the ultrafast MAS frequencies up to 120 kHz is initially discussed, followed by a description of an ensemble of multidimensional NMR pulse sequences, all based on proton detection, that have been developed to obtain in-depth information from dipolar couplings and chemical shift anisotropy (CSA). Simple single channel multidimensional proton NMR experiments could be performed to probe the proximity of protons for structure determination using 1H-1H dipolar couplings and to evaluate the changes in chemical environments as well as the relative orientation to the external magnetic field using proton CSA. Due to the boost in signal sensitivity enabled by proton detection under ultrafast MAS, by virtue of high proton natural abundance and gyromagnetic ratio, proton-detected multidimensional experiments involving low-γ nuclei can now be accomplished within a reasonable time, while the higher dimension also offers additional resolution enhancement. In addition, the application of proton-based ssNMR spectroscopy under ultrafast MAS in various challenging and crystalline systems is also presented. Finally, we briefly discuss the limitations and challenges pertaining to proton-based ssNMR spectroscopy under ultrafast MAS conditions, such as the presence of high-order dipolar couplings, friction-induced sample heating, and limited sample volume. Although there are still a number of challenges that must be circumvented by further developments in radio frequency pulse sequences, MAS probe technology and approaches to prepare NMR-friendly samples, proton-based ssNMR has already gained much popularity in various research domains, especially in proteins where uniform or site-selective deuteration can be relatively easily achieved. In addition, implementation of the recently developed fast data acquisition approaches would also enable further developments in the design and applications of proton-based ultrafast MAS multidimensional ssNMR techniques.
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Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Proteínas/química , Protones , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Using (1)H-based magic angle spinning solid-state NMR spectroscopy, we report an atomistic-level characterization of triglycerides in compact cortical bone. By suppressing contributions from immobile molecules present in bone, we show that a (1)H-based constant-time uniform-sign cross-peak (CTUC) two-dimensional COSY-type experiment that correlates the chemical shifts of protons can selectively detect a mobile triglyceride layer as the main component of small lipid droplets embedded on the surface of collagen fibrils. High sensitivity and resolution afforded by this NMR approach could be potentially utilized to investigate the origin of triglycerides and their pathological roles associated with bone fractures, diseases, and aging.
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Hueso Cortical/química , Espectroscopía de Resonancia Magnética/métodos , Triglicéridos/química , Matriz Extracelular , Resonancia Magnética Nuclear Biomolecular , ProtonesRESUMEN
Degenerative diseases, such as Alzheimer's and prion diseases, as well as type II diabetes, have a pathogenesis associated with protein misfolding, which routes with amyloid formation. Recent strategies for designing small-molecule and polypeptide antiamyloid inhibitors are mainly based on mature fibril structures containing cross ß-sheet structures. In the present study, we have tackled the hypothesis that the rational design of antiamyloid agents that can target native proteins might offer advantageous prospect to design effective therapeutics. Lysozyme amyloid fibrillization was treated with three different peptide fragments derived from lysozyme protein sequence R(107)-R(115). Using low-resolution spectroscopic, high-resolution NMR, and STD NMR-restrained docking methods such as HADDOCK, we have found that these peptide fragments have the capability to affect lysozyme fibril formation. The present study implicates the prospect that these peptides can also be tested against other amyloid-prone proteins to develop novel therapeutic agents.
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Amiloide/química , Muramidasa/química , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Amiloide/ultraestructura , Dicroismo Circular , Microscopía de Fuerza Atómica , Simulación del Acoplamiento Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Mutación Puntual , Unión Proteica , Conformación Proteica , Espectrometría de FluorescenciaRESUMEN
Heteronuclear cross polarization (CP) has been commonly used to enhance the sensitivity of dilute low-γ nuclei in almost all solid-state NMR experiments. However, CP relies on heteronuclear dipolar couplings, and therefore the magnetization transfer efficiency becomes inefficient when the dipolar couplings are weak, as is often the case for mobile components in solids. Here, we demonstrate methods that combine CP with heteronuclear Overhauser effect (referred to as CP-NOE) or with refocused-INEPT (referred to as CP-RINEPT) to overcome the efficiency limitation of CP and enhance the signal-to-noise ratio (S/N) for mobile components. Our experimental results reveal that, compared to the conventional CP, significant S/N ratio enhancement can be achieved for resonances originating from mobile components, whereas the resonance signals associated with rigid groups are not significantly affected due to their long spin-lattice relaxation times. In fact, the S/N enhancement factor is also dependent on the temperature, CP contact time as well as on the system under investigation. Furthermore, we also demonstrate that CP-RINEPT experiment can be successfully employed to independently detect mobile and rigid signals in a single experiment without affecting the data collection time. However, the resolution of CP spectrum obtained from the CP-RINEPT experiment could be slightly compromised by the mandatory use of continuous wave (CW) decoupling during the acquisition of signals from rigid components. In addition, CP-RINEPT experiment can be used for spectral editing utilizing the difference in dynamics of different regions of a molecule and/or different components present in the sample, and could also be useful for the assignment of resonances from mobile components in poorly resolved spectra. Therefore, we believe that the proposed approaches are beneficial for the structural characterization of multiphase and heterogeneous systems, and could be used as a building block in multidimensional solid-state NMR experiments.
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Algoritmos , Espectroscopía de Resonancia Magnética/métodos , Procesamiento de Señales Asistido por Computador , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Marcadores de SpinRESUMEN
Antifreeze proteins (AFPs) are the key biomolecules that enable species to survive under subzero temperature conditions. The physiologically relevant activities of AFPs are based on the adsorption to ice crystals, followed by the inhibition of subsequent crystal layer growth of ice, routed with depression in freezing point in a noncolligative manner. The functional attributes governing the mechanism by which AFPs inhibit freezing of body fluids in bacteria, fungi, plants, and fishes are mainly attributed to their adsorption onto the surface of ice within the physiological system. Importantly, AFPs are also known for their application in cryopreservation of biological samples that might be related to membrane interaction. To date, there is a paucity of information detailing the interaction of AFPs with membrane structures. Here, we focus on elucidating the biophysical properties of the interactions between AFPs and micelle models that mimic the membrane system. Micelle model systems of zwitterionic DPC and negatively charged SDS were utilized in this study, against which a significant interaction is experienced by two AFP molecules, namely, Peptide 1m and wfAFP (the popular AFP sourced from winter flounder). Using low- and high-resolution biophysical characterization techniques, such as circular dichroism (CD) and NMR spectroscopy, a strong evidence for the interactions of these AFPs with the membrane models is revealed in detail and is corroborated by in-depth residue-specific information derived from molecular dynamics simulation. Altogether, these results not only strengthen the fact that AFPs interact actively with membrane systems, but also demonstrate that membrane-associated AFPs are dynamic and capable of adopting a number of conformations rendering fluidity to the system.
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Proteínas Anticongelantes/química , Simulación de Dinámica Molecular , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Conformación Proteica , Marcadores de SpinRESUMEN
Proton NMR spectroscopy in the solid state has recently attracted much attention owing to the significant enhancement in spectral resolution afforded by the remarkable advances in ultrafast magic angle spinning (MAS) capabilities. In particular, proton chemical shift anisotropy (CSA) has become an important tool for obtaining specific insights into inter/intra-molecular hydrogen bonding. However, even at the highest currently feasible spinning frequencies (110-120 kHz), (1)H MAS NMR spectra of rigid solids still suffer from poor resolution and severe peak overlap caused by the strong (1)H-(1)H homonuclear dipolar couplings and narrow (1)H chemical shift (CS) ranges, which render it difficult to determine the CSA of specific proton sites in the standard CSA/single-quantum (SQ) chemical shift correlation experiment. Herein, we propose a three-dimensional (3D) (1)H double-quantum (DQ) chemical shift/CSA/SQ chemical shift correlation experiment to extract the CS tensors of proton sites whose signals are not well resolved along the single-quantum chemical shift dimension. As extracted from the 3D spectrum, the F1/F3 (DQ/SQ) projection provides valuable information about (1)H-(1)H proximities, which might also reveal the hydrogen-bonding connectivities. In addition, the F2/F3 (CSA/SQ) correlation spectrum, which is similar to the regular 2D CSA/SQ correlation experiment, yields chemical shift anisotropic line shapes at different isotropic chemical shifts. More importantly, since the F2/F1 (CSA/DQ) spectrum correlates the CSA with the DQ signal induced by two neighboring proton sites, the CSA spectrum sliced at a specific DQ chemical shift position contains the CSA information of two neighboring spins indicated by the DQ chemical shift. If these two spins have different CS tensors, both tensors can be extracted by numerical fitting. We believe that this robust and elegant single-channel proton-based 3D experiment provides useful atomistic-level structural and dynamical information for a variety of solid systems that possess high proton density.
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Protones , Teoría Cuántica , Anisotropía , Espectroscopía de Resonancia Magnética/normas , Estándares de ReferenciaRESUMEN
Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615-630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution.
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Proteínas 14-3-3/química , Péptidos/química , Proteínas Proto-Oncogénicas c-raf/química , Proteínas 14-3-3/genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Cinética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Péptidos/síntesis química , Fosforilación , Docilidad , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TermodinámicaRESUMEN
While obtaining high-resolution structural details from bone is highly important to better understand its mechanical strength and the effects of aging and disease on bone ultrastructure, it has been a major challenge to do so with existing biophysical techniques. Though solid-state NMR spectroscopy has the potential to reveal the structural details of bone, it suffers from poor spectral resolution and sensitivity. Nonetheless, recent developments in magic angle spinning (MAS) NMR technology have made it possible to spin solid samples up to 110 kHz frequency. With such remarkable capabilities, (1)H-detected NMR experiments that have traditionally been challenging on rigid solids can now be implemented. Here, we report the first application of multidimensional (1)H-detected NMR measurements on bone under ultrafast MAS conditions to provide atomistic-level elucidation of the complex heterogeneous structure of bone. Our investigations demonstrate that two-dimensional (1)H/(1)H chemical shift correlation spectra for bone are obtainable using fp-RFDR (finite-pulse radio-frequency-driven dipolar recoupling) pulse sequence under ultrafast MAS. Our results infer that water exhibits distinct (1)H-(1)H dipolar coupling networks with the backbone and side-chain regions in collagen. These results show the promising potential of proton-detected ultrafast MAS NMR for monitoring structural and dynamic changes caused by mechanical loading and disease in bone.
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Huesos/química , Espectroscopía de Resonancia Magnética , Animales , Bovinos , ProtonesRESUMEN
The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.
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Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Lipopolisacáridos/metabolismo , Enfermedades de las Plantas/prevención & control , Secuencia de Aminoácidos , Antiinfecciosos/química , Antiinfecciosos/toxicidad , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/toxicidad , Calorimetría , Candida/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Virus del Dengue/metabolismo , Escherichia coli/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Lipopolisacáridos/química , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Xanthomonas/efectos de los fármacosRESUMEN
Characterization of the molecular structure and physicochemical solid-state properties of the solid forms of pharmaceutical compounds is a key requirement for successful commercialization as potential active ingredients in drug products. These properties can ultimately have a critical effect on the solubility and bioavailability of the final drug product. Here, the desmotropy of Albendazole forms I and II was investigated at the atomic level. Ultrafast magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy, together with powder X-ray diffraction, thermal analysis, and Fourier transform infrared spectroscopy, were performed on polycrystalline samples of the two solids in order to fully characterize and distinguish the two forms. High-resolution one-dimensional (1)H, (13)C, and (15)N together with two-dimensional (1)H/(1)H single quantum-single quantum, (1)H/(1)H single quantum-double quantum, and (1)H/(13)C chemical shift correlation solid-state NMR experiments under MAS conditions were extensively used to decipher the intramolecular and intermolecular hydrogen bonding interactions present in both solid forms. These experiments enabled the unequivocal identification of the tautomers of each desmotrope. Our results also revealed that both solid forms may be described as dimeric structures, with different intermolecular hydrogen bonds connecting the tautomers in each dimer.
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Albendazol/química , Animales , Antiparasitarios/química , Biofarmacia , Dimerización , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Difracción de Polvo , Espectroscopía Infrarroja por Transformada de Fourier , Estereoisomerismo , TermodinámicaRESUMEN
Reducing the data collection time without affecting the signal intensity and spectral resolution is one of the major challenges for the widespread application of multidimensional nuclear magnetic resonance (NMR) spectroscopy, especially in experiments conducted on complex heterogeneous biological systems such as bone. In most of these experiments, the NMR data collection time is ultimately governed by the proton spin-lattice relaxation times (T1). For over two decades, gadolinium(III)-DTPA (Gd-DTPA, DTPA=Diethylene triamine pentaacetic acid) has been one of the most widely used contrast-enhancement agents in magnetic resonance imaging (MRI). In this study, we demonstrate that Gd-DTPA can also be effectively used to enhance the longitudinal relaxation rates of protons in solid-state NMR experiments conducted on bone without significant line-broadening and chemical-shift-perturbation side effects. Using bovine cortical bone samples incubated in different concentrations of Gd-DTPA complex, the (1)H T1 values were calculated from data collected by (1)H spin-inversion recovery method detected in natural-abundance (13)C cross-polarization magic angle spinning (CPMAS) NMR experiments. Our results reveal that the (1)H T1 values can be successfully reduced by a factor of 3.5 using as low as 10mM Gd-DTPA without reducing the spectral resolution and thus enabling faster data acquisition of the (13)C CPMAS spectra. These results obtained from (13)C-detected CPMAS experiments were further confirmed using (1)H-detected ultrafast MAS experiments on Gd-DTPA doped bone samples. This approach considerably improves the signal-to-noise ratio per unit time of NMR experiments applied to bone samples by reducing the experimental time required to acquire the same number of scans.
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Biopolímeros/análisis , Fémur/química , Gadolinio DTPA/química , Espectroscopía de Resonancia Magnética/métodos , Animales , Polvos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , PorcinosRESUMEN
The intermolecular interaction between mefenamic acid (MFA), a poorly water-soluble nonsteroidal anti-inflammatory drug, and Eudragit EPO (EPO), a water-soluble polymer, is investigated in their supersaturated solution using high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy. The stable supersaturated solution with a high MFA concentration of 3.0 mg/mL is prepared by dispersing the amorphous solid dispersion into a d-acetate buffer at pH 5.5 and 37 °C. By virtue of MAS at 2.7 kHz, the extremely broad and unresolved (1)H resonances of MFA in one-dimensional (1)H NMR spectrum of the supersaturated solution are well-resolved, thus enabling the complete assignment of MFA (1)H resonances in the aqueous solution. Two-dimensional (2D) (1)H/(1)H nuclear Overhauser effect spectroscopy (NOESY) and radio frequency-driven recoupling (RFDR) under MAS conditions reveal the interaction of MFA with EPO in the supersaturated solution at an atomic level. The strong cross-correlations observed in the 2D (1)H/(1)H NMR spectra indicate a hydrophobic interaction between the aromatic group of MFA and the backbone of EPO. Furthermore, the aminoalkyl group in the side chain of EPO forms a hydrophilic interaction, which can be either electrostatic or hydrogen bonding, with the carboxyl group of MFA. We believe these hydrophobic and hydrophilic interactions between MFA and EPO molecules play a key role in the formation of this extremely stable supersaturated solution. In addition, 2D (1)H/(1)H RFDR demonstrates that the molecular MFA-EPO interaction is quite flexible and dynamic.
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Antiinflamatorios no Esteroideos/química , Espectroscopía de Resonancia Magnética , Ácido Mefenámico/química , Ácidos Polimetacrílicos/química , SolucionesRESUMEN
Solid-state (magic-angle spinning) NMR spectroscopy is a useful tool for obtaining structural information on bone organic and mineral components and synthetic model minerals at the atomic-level. Raman and 31P NMR spectral parameters were investigated in a series of synthetic B-type carbonated apatites (CAps). Inverse 31P NMR linewidth and inverse Raman PO43- ν1 bandwidth were both correlated with powder XRD c-axis crystallinity over the 0.3-10.3 wt% CO32- range investigated. Comparison with bone powder crystallinities showed agreement with values predicted by NMR and Raman calibration curves. Carbonate content was divided into two domains by the 31P NMR chemical shift frequency and the Raman phosphate ν1 band position. These parameters remain stable except for an abrupt transition at 6.5 wt% carbonate, a composition which corresponds to an average of one carbonate per unit cell. This near-binary distribution of spectroscopic properties was also found in AFM-measured particle sizes and Ca/P molar ratios by elemental analysis. We propose that this transition differentiates between two charge-balancing ion-loss mechanisms as measured by Ca/P ratios. These results define a criterion for spectroscopic characterization of B-type carbonate substitution in apatitic minerals.