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Tardigrades are unique micro-organisms with a high tolerance to desiccation. The protection of their cells against desiccation involves tardigrade-specific proteins, which include the so-called cytoplasmic abundant heat soluble (CAHS) proteins. As a first step towards the design of peptides capable of mimicking the cytoprotective properties of CAHS proteins, we have synthesized several model peptides with sequences selected from conserved CAHS motifs and investigated to what extent they exhibit the desiccation-induced structural changes of the full-length proteins. Using circular dichroism spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations, we have found that the CAHS model peptides are mostly disordered, but adopt a more α $$ \alpha $$ -helical structure upon addition of 2,2,2-trifluoroethanol, which mimics desiccation. This structural behavior is similar to that of full-length CAHS proteins, which also adopt more ordered conformations upon desiccation. We also have investigated the surface activity of the peptides at the air/water interface, which also mimics partial desiccation. Interestingly, sum-frequency generation spectroscopy shows that all model peptides are surface active and adopt a helical structure at the air/water interface. Our results suggest that amino acids with high helix-forming propensities might contribute to the propensity of these peptides to adopt a helical structure when fully or partially dehydrated. Thus, the selected sequences retain part of the CAHS structural behavior upon desiccation, and might be used as a basis for the design of new synthetic peptide-based cryoprotective materials.
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Simulación de Dinámica Molecular , Péptidos , Tardigrada , Tardigrada/química , Animales , Péptidos/química , Estructura Secundaria de Proteína , Secuencia de AminoácidosRESUMEN
ß-Lactoglobulin is a main allergen in cow's milk; its allergenicity is strongly impacted by processing. To understand heat-induced epitope-specific effects, the present study analyzed regiospecific conformational changes of heated native ß-lactoglobulin variant A (BLG-A). Complementary fluorescence spectroscopy methods indicated two denaturation phases comprising minor sequential conformational changes (25-75 °C) and complete transitions (80-90 °C). Regioselective conformational changes of BLG-A in the native state (25 °C), sequential (70 °C) and complete transition (90 °C) were determined by quantitative liquid chromatography-mass spectrometry analysis of chemical labeling kinetics covering 14 lysine residues and the N-terminus. Conformational changes in two phases were observed for N-terminus, K8 (both N-terminal chain), K60 (ß-sheet C), K75 (ß-sheet D), K77 (DE loop), K83 (ß-sheet E), K100 and K101 (FG loop). The residues K14 (ß-sheet A1), K47 (ß-sheet B), K69, K70 (both ß-sheet D), and K91 (ß-sheet F) were not involved in conformational changes.
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HIV-1 can rapidly infect the brain upon initial infection, establishing latent reservoirs that induce neuronal damage and/or death, resulting in HIV-Associated Neurocognitive Disorder. Though anti-HIV-1 antiretrovirals (ARVs) suppress viral load, the blood-brain barrier limits drug access to the brain, largely because of highly expressed efflux proteins like P-glycoprotein (P-gp). While no FDA-approved P-gp inhibitor currently exists, HIV-1 protease inhibitors show promise as partial P-gp inhibitors, potentially enhancing drug delivery to the brain. Herein, we employed docking and molecular dynamics simulations to elucidate key differences in P-gp's interactions with several antiretrovirals, including protease inhibitors, with known inhibitory or substrate-like behaviors towards P-gp. Our results led us to hypothesize new mechanistic details of small-molecule efflux by and inhibition of P-gp, where the "Lower Pocket" in P-gp's transmembrane domain serves as the primary initial site for small-molecule binding. Subsequently, this pocket merges with the more traditionally studied drug binding site-the "Upper Pocket"-thus funneling small-molecule drugs, such as ARVs, towards the Upper Pocket for efflux. Furthermore, our results reinforce the understanding that both binding energetics and changes in protein dynamics are crucial in discerning small molecules as non-substrates, substrates, or inhibitors of P-gp. Our findings indicate that interactions between P-gp and inhibitory ARVs induce bridging of transmembrane domain helices, impeding P-gp conformational changes and contributing to the inhibitory behavior of these ARVs. Overall, insights gained in this study could serve to guide the design of future P-gp-targeting therapeutics for a wide range of pathological conditions and diseases, including HIV-1.
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Transglutaminase (TGase)-catalyzed crosslinking has gained substantial traction as a novel strategy for reducing allergenic risk in food proteins, particularly within the realm of hypoallergenic food production. This study explored the impact of TGase crosslinking on conformational changes in a binary protein system composed of soy protein isolate (SPI) and sodium caseinate (SC) at varying mass ratios (10:0, 7:3, 5:5, 3:7 (w/w)). Specifically, the immunoglobulin E (IgE) binding capacity of soy proteins within this system was examined. Prolonged TGase crosslinking (ranging from 0 h to 15 h) resulted in a gradual reduction in IgE reactivity across all SPI-SC ratios, with the order of IgE-binding capability as follows: SPI > SPI5-SC5 > SPI7-SC3 > SPI3-SC7. These alterations in protein conformation following TGase crosslinking, as demonstrated by variable intrinsic fluorescence, altered surface hydrophobicity, increased ultraviolet absorption and reduced free sulfhydryl content, were identified as the underlying causes. Additionally, ionic bonds were found to play a significant role in maintaining the structure of the dual-protein system after crosslinking, with hydrophobic forces and hydrogen bonds serving as supplementary forces. Generally, the dual-protein system may exhibit enhanced efficacy in reducing the allergenicity of soy protein.
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Inmunoglobulina E , Conformación Proteica , Proteínas de Soja , Transglutaminasas , Transglutaminasas/química , Transglutaminasas/metabolismo , Proteínas de Soja/química , Proteínas de Soja/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Reactivos de Enlaces Cruzados/química , Interacciones Hidrofóbicas e Hidrofílicas , Humanos , Caseínas/química , Caseínas/metabolismo , Caseínas/inmunologíaRESUMEN
Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.
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Detergentes , Conformación Proteica , Intercambiador de Sodio-Calcio , Detergentes/química , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/genética , Iones/química , Nanoestructuras/química , Solubilidad , Animales , Espectroscopía de Resonancia Magnética/métodosRESUMEN
In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7â¯Å and 2.0â¯Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis.
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The interaction between nucleotide molecules and lipid molecules plays important roles in cell activities, but the molecular mechanism is very elusive. In the present study, a small but noticeable interaction between the negatively charged phosphatidylethanolamine (PE) and Guanosine monophosphate (GMP) molecules was observed from the PE monolayer at the air/water interface. As shown by the sum frequency generation (SFG) spectra and Pi-A isotherm of the PE monolayer, the interaction between the PE and GMP molecules imposes very small changes to the PE molecules. However, the Brewster angle microscopy (BAM) technique revealed that the assembly conformations of PE molecules are significantly changed by the adsorption of GMP molecules. By comparing the SFG spectra of PE monolayers after the adsorption of GMP, guanosine and guanine, it is also shown that the hydrogen bonding effect plays an important role in the nucleotide-PE interactions. These results provide fundamental insight into the structure changes during the nucleotide-lipid interaction, which may shed light on the molecular mechanism of viral infection, DNA drug delivery, and cell membrane curvature control in the brain or neurons.
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Guanosina Monofosfato , Fosfatidiletanolaminas , Fosfatidiletanolaminas/química , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Adsorción , Propiedades de Superficie , Microscopía , Enlace de Hidrógeno , Agua/químicaRESUMEN
Intrinsically disordered protein regions (IDPRs) are pivotal in regulation of transcription and facilitation of signal transduction. Because of their multiple conformational states of structure, characterizing the highly flexible structures of IDPRs becomes challenging. Herein, we employed the wild-type (WT) aerolysin nanopore as a real-time biosensor for identification and monitoring of long peptides containing IDPRs. This sensor successfully identified three intrinsically disordered peptides, with the lengths up to 43 amino acids, by distinguishing the unique signatures of blockade current and duration time. The analysis of the binding constant revealed that interactions between the nanopore and peptides are critical for peptide translocation, which suggests that mechanisms beyond mere volume exclusion. Furthermore, we were able to compare the conformational stabilities of various IDPRs by examining the detailed current traces of blockade events. Our approach can detect the conformational changes of IDPR in a confined nanopore space. These insights broaden the understanding of peptide structural changes. The nanopore biosensor showed the potential to study the conformations change of IDPRs, IDPRs transmembrane interactions, and protein drug discovery.
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Angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated ACE expression in tissues (which is generally reflected by blood ACE levels) is associated with an increased risk of cardiovascular diseases. Elevated blood ACE is also a marker for granulomatous diseases. Decreased blood ACE activity is becoming a new risk factor for Alzheimer's disease. We applied our novel approach-ACE phenotyping-to characterize pairs of tissues (lung, heart, lymph nodes) and serum ACE in 50 patients. ACE phenotyping includes (1) measurement of ACE activity with two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of these substrates (ZPHL/HHL ratio); (3) determination of ACE immunoreactive protein levels using mAbs to ACE; and (4) ACE conformation with a set of mAbs to ACE. The ACE phenotyping approach in screening format with special attention to outliers, combined with analysis of sequencing data, allowed us to identify patient with a unique ACE phenotype related to decreased ability of inhibition of ACE activity by albumin, likely due to competition with high CCL18 in this patient for binding to ACE. We also confirmed recently discovered gender differences in sialylation of some glycosylation sites of ACE. ACE phenotyping is a promising new approach for the identification of ACE phenotype outliers with potential clinical significance, making it useful for screening in a personalized medicine approach.
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BACKGROUND: Food allergies are a growing concern worldwide, with soy proteins being important allergens that are widely used in various food products. This study investigated the potential of transglutaminase (TGase) and lactic acid bacteria (LAB) treatments to modify the allergenicity and structural properties of soy protein isolate (SPI), aiming to develop safer soy-based food products. RESULTS: Treatment with TGase, LAB or their combination significantly reduced the antibody reactivity of ß-conglycinin and the immunoglobulin E (IgE) binding capacity of soy protein, indicating a decrease in allergenicity. TGase treatment led to the formation of high-molecular-weight aggregates, suggesting protein crosslinking, while LAB treatment resulted in partial protein hydrolysis. These structural changes were confirmed by Fourier transform infrared spectroscopy, which showed a decrease in ß-sheet content and an increase in random coil and ß-turn contents. In addition, changes in intrinsic fluorescence and ultraviolet spectroscopy were also observed. The alterations in protein interaction and the reduction in free sulfhydryl groups highlighted the extensive structural modifications induced by these treatments. CONCLUSION: The synergistic application of TGase and LAB treatments effectively reduced the allergenicity of SPI through significant structural modifications. This approach not only diminished antibody reactivity of ß-conglycinin and IgE binding capacity of soy protein but also altered the protein's primary, secondary and tertiary structures, suggesting a comprehensive alteration of SPI's allergenic potential. These findings provide a promising strategy for mitigating food allergy concerns and lay the foundation for future research on food-processing techniques aimed at allergen reduction. © 2024 Society of Chemical Industry.
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The interaction of biomacromolecules with each other or with the ligands is essential for biological activity. In this context, the molecular recognition of bovine serum albumin (BSA) with 4-(Benzo[1,3]dioxol-5-yloxymethyl)-7-hydroxy-chromen-2-one (4BHC) is explored using multispectroscopic and computational techniques. UV-Vis spectroscopy helped in predicting the conformational variations in BSA. Using fluorescence spectroscopy, the quenching behaviour of the fluorophore upon interaction with the ligand is examined, which is found to be a static type of quenching; fluorescence lifetime studies further verify this. The binding constant is discovered to be in the range of 104 M-1, which indicates the moderate type of association that results in reversible binding, where the transport and release of ligands in the target tissue takes place. Fourier-transform infrared spectroscopy (FT-IR) measurements validate the secondary structure conformational changes of BSA after complexing with 4BHC. The thermodynamic factors obtained through temperature-dependent fluorescence studies suggest that the dominant kind of interaction force is hydrophobic in nature, and the interaction process is spontaneous. The alterations in the surrounding microenvironment of the binding site and conformational shifts in the structure of the protein are studied through 3D fluorescence and synchronous fluorescence studies. Molecular docking and molecular dynamics (MD) simulations agree with experimental results and explain the structural stability throughout the discussion. The outcomes might have possible applications in the field of pharmacodynamics and pharmacokinetics.
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Cumarinas , Simulación del Acoplamiento Molecular , Albúmina Sérica Bovina , Espectrometría de Fluorescencia , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Animales , Bovinos , Cumarinas/química , Cumarinas/metabolismo , Termodinámica , Unión Proteica , Colorantes Fluorescentes/química , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , Sitios de Unión , Simulación por ComputadorRESUMEN
Cyclodextrins (CDs) can enhance the stability and bioavailability of pharmaceutical compounds by encapsulating them within their cavities. This study utilized molecular dynamics simulations to investigate the interaction mechanisms between hydrocortisone (HC) and various methylated CD derivatives. The results reveal that the loading of HC into CD cavities follows different mechanisms depending on the degree and position of methylation. Loading into ßCD and 6-MeßCD was more complete, with the hydroxyl groups of HC facing the primary hydroxyl rim (PHR) and the ketone side facing the secondary hydroxyl rim (SHR). In contrast, 2,3-D-MeßCD and 2,6-D-MeßCD showed a different loading mechanism, with the ketone side facing the PHR and the hydroxyl groups facing the SHR. The root mean square fluctuation (RMSF) analysis demonstrated that methylation increases the flexibility of CD heavy atoms, with 3-MeßCD and 2,3-D-MeßCD exhibiting the highest flexibility. However, upon inclusion of HC, 3-MeßCD, 2,3-D-MeßCD, 2-MeßCD, and 6-MeßCD showed a significant reduction in flexibility, suggesting a more rigid structure that effectively retains HC within their cavities. The radial distribution function revealed a significant reduction in the number of water molecules within the innermost layer of the methylated CD cavities, particularly in TMeßCD, indicating a decrease in polarity. The presence of HC led to the release of high-energy water molecules, creating more favorable conditions for HC loading. Conformational analysis showed that methylation caused a partial decrease in the area of the PHR, a significant decrease in the area of the middle rim, and a notable decrease in the area of the SHR. The loading of HC increased the area of the PHR in most derivatives, with the most pronounced increase observed in 2,6-D-MeßCD and 6-MeßCD. The analysis of interaction energies and binding free energies demonstrated that the binding of HC to methylated CD derivatives is thermodynamically more favorable than to ßCD, with the strongest association observed for 6-MeßCD, 2-MeßCD, and 2,3-D-MeßCD.
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Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.
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Integrinas , Talina , Animales , Humanos , Ratones , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Adhesión Celular , Células CHO , Cricetulus , Integrinas/metabolismo , Integrinas/química , Ligandos , Unión Proteica , Conformación Proteica , Transducción de Señal , Imagen Individual de Molécula , Talina/metabolismo , Talina/químicaRESUMEN
The unique prolyl isomerase Pin1 binds to and catalyzes cis-trans conformational changes of specific Ser/Thr-Pro motifs after phosphorylation, thereby playing a pivotal role in regulating the structure and function of its protein substrates. In particular, Pin1 activity regulates the affinity of a substrate for E3 ubiquitin ligases, thereby modulating the turnover of a subset of proteins and coordinating their activities after phosphorylation in both physiological and disease states. In this review, we highlight recent advancements in Pin1-regulated ubiquitination in the context of cancer and neurodegenerative disease. Specifically, Pin1 promotes cancer progression by increasing the stabilities of numerous oncoproteins and decreasing the stabilities of many tumor suppressors. Meanwhile, Pin1 plays a critical role in different neurodegenerative disorders via the regulation of protein turnover. Finally, we propose a novel therapeutic approach wherein the ubiquitin-proteasome system can be leveraged for therapy by targeting pathogenic intracellular targets for TRIM21-dependent degradation using stereospecific antibodies.
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Peptidilprolil Isomerasa de Interacción con NIMA , Proteolisis , Ubiquitinación , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Conformación Proteica , Animales , Neoplasias/metabolismo , Neoplasias/patología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
ATP has recently been reconsidered as a molecule with functional properties which go beyond its recognized role of the energetic driver of the cell. ATP has been described as an allosteric modulator as well as a biological hydrotrope with anti-aggregation properties in the crowded cellular environment. The role of ATP as a modulator of the homeostasis of the neurotrophins (NTs), a growth factor protein family whose most known member is the nerve growth factor (NGF), has been investigated. The modulation of NTs by small endogenous ligands is still a scarcely described area, with few papers reporting on the topic, and very few reports on the molecular determinants of these interactions. However, a detailed atomistic description of the NTs interaction landscape is of urgent need, aiming at the identification of novel molecules as potential therapeutics and considering the wide range of potential pharmacological applications for NGF and its family members. This mini-review will focus on the unique cartography casting the interactions of the endogenous ligand ATP, in the interaction with NGF as well as with its precursor proNGF. These interactions revealed interesting features of the ATP binding and distinct differences in the binding mode between the highly structured mature NGF and its precursor, proNGF, which is characterized by an intrinsically unstructured domain. The overview on the recent available data will be presented, together with the future perspectives on the field.
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Adenosina Trifosfato , Factor de Crecimiento Nervioso , Unión Proteica , Factor de Crecimiento Nervioso/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Animales , Precursores de Proteínas/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/química , Ligandos , Sitios de UniónRESUMEN
In binding free energy calculations, simulations must sample all relevant conformations of the system in order to obtain unbiased results. For instance, different ligands can bind to different metastable states of a protein, and if these protein conformational changes are not sampled in relative binding free energy calculations, the contribution of these states to binding is not accounted for and thus calculated binding free energies are inaccurate. In this work, we investigate the impact of different beta-sectretase 1 (BACE1) protein conformations obtained from x-ray crystallography on the binding of BACE1 inhibitors. We highlight how these conformational changes are not adequately sampled in typical molecular dynamics simulations. Furthermore, we show that insufficient sampling of relevant conformations induces substantial error in relative binding free energy calculations, as judged by a variation in calculated relative binding free energies up to 2 kcal/mol depending on the starting protein conformation. These results emphasize the importance of protein conformational sampling and pose this BACE1 system as a challenge case for further method development in the area of enhanced protein conformational sampling, either in combination with binding calculations or as an endpoint correction.
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Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Termodinámica , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Humanos , Cristalografía por Rayos X , LigandosRESUMEN
The formulation of drug with improved bioavailability is always challenging and indispensable in the field of pharmaceutics. The control of intermolecular interactions via crystal engineering approach and solid-state molecular recognition results in the formation of active drug molecules with modulated pharmacological benefits. Therefore, with the aim to improve the solubility and dissolution rate of the drug chlorpropamide (CPA), the mechanochemical liquid-assisted grinding (LAG) of the drug with several pharmaceutically accepted excipients was performed. This contributed to the discovery of six novel solid phases, namely salts, salt cocrystals and salt cocrystal hydrateâthe salt of CPA with 3, 4-diaminopyridine (DAP); salt and salt cocrystal (SC) polymorph (Zâ³=3) with 1, 4-diazabicyclo [2.2.2] octane (DABCO); a salt, SC polymorph (Zâ³=9), and a SC hydrate (Zâ³=9) with piperazine (PIP). The formation of these salts and salt cocrystals are mainly guided by the strong hydrogen bonds with tunable strength having high electrostatic contribution. This attractive interaction brings the donor and the acceptor atoms close to each other for a facile proton transfer. Furthermore, the conformational constraints on the drug molecules, provided by the excipients via strong and directional hydrogen bonds, are quite impressive as this leads to the identification and characterization of "new conformational isomers" for the CPA molecules. The new crystalline phases exhibit enhanced intrinsic dissolution rate in comparison to that of the pure drug, the magnitude being 7, 131, and 120 folds for CPADAP, CPADABCO_II, and CPAPIP_III, respectively. Furthermore, it is interesting to note that the order of solubility is enhanced by 2.7-, 3-, and 7-fold, respectively, for the abovementioned salts. This also mirrors the trends in the magnitude of the binding energy, the higher magnitude being reflected in the lower solubility. Additionally, the in vivo experiments performed in SD rats results in the enhancement of the magnitude of the pharmacokinetic properties, when compared to the pristine drug. The concentration of the drug in CPADABCO_II and CPAPIP_III formulations exhibits 6- and 4-fold increments, respectively. Indeed, these results corroborate to the trends observed in the structural characterization, intermolecular energy calculations, solubility, and in vitro dissolution assessments.
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Clorpropamida , Cristalización , Enlace de Hidrógeno , Sales (Química) , Solubilidad , Cristalización/métodos , Sales (Química)/química , Clorpropamida/química , Química Farmacéutica/métodos , Excipientes/química , Composición de Medicamentos/métodos , Animales , Ratas , Disponibilidad BiológicaRESUMEN
X-ray crystallography and cryo-electron microscopy have enabled the determination of structures of numerous viruses at high resolution and have greatly advanced the field of structural virology. These structures represent only a subset of snapshot end-state conformations, without describing all conformational transitions that virus particles undergo. Allostery plays a critical role in relaying the effects of varied perturbations both on the surface through environmental changes and protein (receptor/antibody) interactions into the genomic core of the virus. Correspondingly, allostery carries implications for communicating changes in genome packaging to the overall stability of the virus particle. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) of whole viruses is a powerful probe for uncovering virus allostery. Here we critically discuss advancements in understanding virus dynamics by HDXMS with single particle cryo-EM and computational approaches.
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Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Virión , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Virión/química , Virión/metabolismo , Regulación Alostérica , Amidas/química , Virus/química , Virus/metabolismo , Microscopía por Crioelectrón/métodos , Espectrometría de Masas/métodos , Medición de Intercambio de DeuterioRESUMEN
Interferon-gamma-inducible large GTPases, hGBPs, possess antipathogenic and antitumor activities in human cells. Like hGBP1, its closest homolog, hGBP3 has two domains; an N-terminal catalytic domain and a C-terminal helical domain, connected by an intermediate region. The biochemical function of this protein and the role of its domains in substrate hydrolysis have not yet been investigated. Here, we report that while hGBP3 can produce both GDP and GMP, GMP is the minor product, 30% (unlike 85% in hGBP1), indicating that hGBP3 is unable to produce enhanced GMP. To understand which domain(s) are responsible for this deficiency, we created hGBP3 truncated variants. Surprisingly, GMP production was similar upon deletion of the helical domain, suggesting that in contrast to hGBP1, the helical domain of hGBP3 cannot stimulate the second phosphate cleavage of GTP. We conducted computational and solution studies to understand the underlying basis. We found that the regulatory residue W79, present in the catalytic domain, forms an H-bond with the backbone carbonyl of K76 (located in the catalytic loop) of the substrate-bound hGBP3. However, after gamma-phosphate cleavage of GTP, the W79-containing region does not undergo a conformational change, failing to redirect the catalytic loop toward the beta-phosphate. This is necessary for efficient GMP formation because hGBP homologs utilize the same catalytic residue for both phosphate cleavages. We suggest that the lack of specific interdomain contacts mediated by the helical domain prevents the catalytic loop movement, resulting in reduced GMP formation. These findings may provide insight into how hGBP3 contributes to immunity.
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Dominio Catalítico , Proteínas de Unión al GTP , Guanosina Trifosfato , Fosfatos , Humanos , Dominio Catalítico/genética , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Fosfatos/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismoRESUMEN
ABCG2 is an important ATP-binding cassette transporter impacting the absorption and distribution of over 200 chemical toxins and drugs. ABCG2 also reduces the cellular accumulation of diverse chemotherapeutic agents. Acquired somatic mutations in the phylogenetically conserved amino acids of ABCG2 might provide unique insights into its molecular mechanisms of transport. Here, we identify a tumor-derived somatic mutation (Q393K) that occurs in a highly conserved amino acid across mammalian species. This ABCG2 mutant seems incapable of providing ABCG2-mediated drug resistance. This was perplexing because it is localized properly and retained interaction with substrates and nucleotides. Using a conformationally sensitive antibody, we show that this mutant appears "locked" in a non-functional conformation. Structural modeling and molecular dynamics simulations based on ABCG2 cryo-EM structures suggested that the Q393K interacts with the E446 to create a strong salt bridge. The salt bridge is proposed to stabilize the inward-facing conformation, resulting in an impaired transporter that lacks the flexibility to readily change conformation, thereby disrupting the necessary communication between substrate binding and transport.