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The hallmark of amyloidosis, such as Alzheimer's disease and Parkinson's disease, is the deposition of amyloid fibrils in various internal organs. The onset of the disease is related to the strength of cytotoxicity caused by toxic amyloid species. Furthermore, amyloid fibrils show polymorphism, where some types of fibrils are cytotoxic while others are not. It is thus essential to understand the molecular mechanism of cytotoxicity, part of which is caused by the interaction between amyloid polymorphic fibrils and cell membranes. Here, using amyloid polymorphs of hen egg white lysozyme, which is associated with hereditary systemic amyloidosis, showing different levels of cytotoxicity and liposomes of DMPC and DMPG, changes in the secondary structure of the polymorphs and the structural state of phospholipid membranes caused by the interaction were investigated using vacuum-ultraviolet circular dichroism (VUVCD) and Laurdan fluorescence measurements, respectively. Analysis has shown that the more cytotoxic polymorph increases the antiparallel ß-sheet content and causes more disorder in the membrane structure while the other less cytotoxic polymorph shows the opposite structural changes and causes less structural disorder in the membrane. These results suggest a close correlation between the structural properties of amyloid fibrils and the degree of structural disorder of phospholipid membranes, both of which are involved in the fundamental process leading to amyloid cytotoxicity.
Assuntos
Amiloide , Dicroísmo Circular , Muramidase , Fosfolipídeos , Muramidase/química , Muramidase/metabolismo , Amiloide/química , Fosfolipídeos/química , Animais , Estrutura Secundária de Proteína , Dimiristoilfosfatidilcolina/química , Fosfatidilgliceróis/química , Lipossomos/química , Galinhas , VácuoRESUMO
One of the routes for adaptation to extreme environments is via remodeling of cell membrane structure, composition, and biophysical properties rendering a functional membrane. Collective studies suggest some form of membrane feedback in mycobacterial species that harbor complex lipids within the outer and inner cell wall layers. Here, we study the homeostatic membrane landscape of mycobacteria in response to high hydrostatic pressure and temperature triggers using high pressure fluorescence, mass and infrared spectroscopies, NMR, SAXS, and molecular dynamics simulations. Our findings reveal that mycobacterial membrane possesses unique and lipid-specific pressure-induced signatures that attenuate progression to highly ordered phases. Both inner and outer membrane layers exhibit phase coexistence of nearly identical lipid phases keeping residual fluidity over a wide range of temperature and pressure, but with different sensitivities. Lipidomic analysis of bacteria grown under pressure revealed lipidome remodeling in terms of chain length, unsaturation, and specific long-chained characteristic mycobacterial lipids, rendering a fluid bacterial membrane. These findings could help understand how bacteria may adapt to a broad spectrum of harsh environments by modulating their lipidome to select lipids that enable the maintenance of a fluid functional cell envelope.
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Membrana Celular , Fluidez de Membrana , Simulação de Dinâmica Molecular , Membrana Celular/química , Membrana Celular/metabolismo , Temperatura , Parede Celular/metabolismo , Parede Celular/química , Adaptação Fisiológica , Pressão Hidrostática , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismoRESUMO
NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1H, 15N and 13C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners.
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Ressonância Magnética Nuclear Biomolecular , Humanos , Sequência de Aminoácidos , Estrutura Secundária de Proteína , Isótopos de NitrogênioRESUMO
NCYM, a Homininae-specific oncoprotein, is the first de novo gene product experimentally shown to have oncogenic functions. NCYM stabilizes MYCN and ß-catenin via direct binding and inhibition of GSK3ß and promotes cancer progression in various tumors. Thus, the identification of compounds that binds to NCYM and structural characterization of the complex of such compounds with NCYM are required to deepen our understanding of the molecular mechanism of NCYM function and eventually to develop anticancer drugs against NCYM. In this study, the DNA aptamer that specifically binds to NCYM and enhances interaction between NCYM and GSK3ß were identified for the first time using systematic evolution of ligands by exponential enrichment (SELEX). The structural properties of the complex of the aptamer and NCYM were investigated using atomic force microscopy (AFM) in combination with truncation and mutation of DNA sequence, pointing to the regions on the aptamer required for NCYM binding. Further analysis was carried out by small-angle X-ray scattering (SAXS). Structural modeling based on SAXS data revealed that when isolated, NCYM shows high flexibility, though not as a random coil, while the DNA aptamer exists as a dimer in solution. In the complex state, models in which NCYM was bound to a region close to an edge of the aptamer reproduced the SAXS data. Therefore, using a combination of SELEX, AFM, and SAXS, the present study revealed the structural properties of NCYM in its functionally active form, thus providing useful information for the possible future design of novel anti-cancer drugs targeting NCYM.
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Thermal fluctuations of proteins at the ps-ns timescales are important for their functions and have extensively been studied using quasi-elastic neutron scattering (QENS). In general, the QENS spectra of proteins are analyzed in terms of two populations of atoms, i.e., the immobile fraction of atoms, the motions of which are too slow to be resolved within the instrumental energy resolution employed, and the mobile fraction of atoms, from which the average amplitude and frequency of protein atomic motions are characterized. On the other hand, it has been shown using molecular dynamics simulations that atomic motions are gradually enhanced as going from the protein core to the protein surface. Therefore, it is essential to further deconvolute the mobile fraction of atoms to study in detail the dynamical behavior of proteins. Here, an improved analytical model using QENS to deconvolute the mobile fraction of atoms into two populations of atoms, i.e., atoms with high mobility (HM atoms) and those with low mobility (LM atoms), is proposed. It was found that both the HM atoms and the LM atoms showed gradually enhanced dynamics with an increase in temperature even though any temperature-dependent terms are not included in the model. The presented model yields physically reasonable values for dynamical parameters and hence its future application will be useful to understand the molecular mechanism of various protein functions where atoms with higher mobility on or close to the protein surface play a crucial role.
Assuntos
Simulação de Dinâmica Molecular , Proteínas , TemperaturaRESUMO
Incoherent neutron scattering (iNS) is one of the most powerful techniques to study the dynamical behavior of bio-macromolecules such as proteins and lipid molecules or whole cells. This technique has widely been used to elucidate the fundamental aspects of molecular motions that manifest in the bio-macromolecules in relation to their intrinsic molecular properties and biological functions. Furthermore, in the last decade, iNS studies focusing on a possible relationship between molecular dynamics and biological malfunctions, i.e., human diseases and disorders, have gained importance. In this review, we summarize recent iNS studies on pathologically relevant proteins and lipids and discuss how the findings are of importance to elucidate the molecular mechanisms of human diseases and disorders that each study targets. Since some diseases such as amyloidosis have become more relevant in the aging society, research in this field will continue to develop further and be more important in the current increasing trend for longevity worldwide.
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Small-angle scattering is a powerful technique to obtain structural information on biomacromolecules in aqueous solution at the sub-nanometer and nanometer length scales. It provides the sizes and overall shapes of the scattering particles. While small-angle X-ray scattering (SAXS) has often been used for structural analysis of a single-component system, small-angle neutron scattering (SANS) has been used to reveal the internal organization of a multicomponent system such as protein-protein and protein-DNA complexes. This is due to the fact that the neutron scattering length is largely different between hydrogen and deuterium, and thus it allows to make a specific component in complexes "invisible" to neutrons by changing the H2O/D2O ratio in the solvent with or without molecular deuteration. In this chapter, we describe a method to characterize the biomolecular structures using SANS and SAXS, in particular, focusing on fibrillar proteins such as bacterial amyloids and their complexes with nucleic acids.
Assuntos
Difração de Nêutrons , Nêutrons , Proteínas Amiloidogênicas , DNA , Difração de Nêutrons/métodos , Espalhamento a Baixo Ângulo , Difração de Raios X , Raios XRESUMO
X-ray/neutron fiber diffraction and small-angle X-ray/neutron scattering are widely used to investigate the molecular structure of fibrous proteins, including amyloid fibrils. However, there is sometimes confusion between these two techniques despite the fact that sample conditions and the content of the information obtained are not the same. In this brief chapter, we present the differences in sample conditions between these two methods, and their effects on experimentally obtained diffraction or scattering patterns, emphasizing the degree of disorder in the samples.
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Amiloide , Nêutrons , Amiloide/química , Difração de Nêutrons , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Modern phospholipid membranes are known to be in a functional, physiological state, corresponding to the liquid crystalline phase, only under very precise external conditions. The phase is characterised by specific lipid motions, which seem mandatory to permit sufficient flexibility and stability for the membrane. It can be assumed that similar principles hold for proto-membranes at the origin of life although they were likely composed of simpler, single chain fatty acids and alcohols. In the present study we investigated molecular motions of four types of model membranes to shed light on the variations of dynamics and structure from low to high temperature as protocells might have existed close to hot vents. We find a clear hierarchy among the flexibilities of the samples, where some structural parameters seem to depend on the lipid type used while others do not.
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Células Artificiais , Fosfolipídeos , Temperatura Alta , Bicamadas Lipídicas/química , Movimento (Física) , Fosfolipídeos/química , TemperaturaRESUMO
In accompanying papers [Bicout et al., BioRxiv https://doi.org/10.1101/2021.09.21.461198 (2021); Cissé et al., BioRxiv https://doi.org/10.1101/2022.03.30.486370 (2022)], a new model called Matryoshka model has been proposed to describe the geometry of atomic motions in phospholipid molecules in bilayers and multilamellar vesicles based on their quasielastic neutron scattering (QENS) spectra. Here, in order to characterize the relaxational aspects of this model, the energy widths of the QENS spectra of the samples were analyzed first in a model-free way. The spectra were decomposed into three Lorentzian functions, which are classified as slow, intermediate, and fast motions depending on their widths. The analysis provides the diffusion coefficients, residence times, and geometrical parameters for the three classes of motions. The results corroborate the parameter values such as the amplitudes and the mobile fractions of atomic motions obtained by the application of the Matryoshka model to the same samples. Since the current analysis was carried out independently of the development of the Matryoshka model, the present results enhance the validity of the model. The model will serve as a powerful tool to decipher the dynamics of lipid molecules not only in model systems, but also in more complex systems such as mixtures of different kinds of lipids or natural cell membranes.
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Difração de Nêutrons , Nêutrons , Difusão , Movimento (Física) , Difração de Nêutrons/métodos , FosfolipídeosRESUMO
Fluid lipid bilayers are the building blocks of biological membranes. Although there is a large amount of experimental data using incoherent quasi-elastic neutron scattering (QENS) techniques to study membranes, very little theoretical works have been developed to study the local dynamics of membranes. The main objective of this work is to build a theoretical framework to study and describe the local dynamics of lipids and derive analytical expressions of intermediate scattering functions (ISF) for QENS. As results, we developed the dynamical Matryoshka model which describes the local dynamics of lipid molecules in membrane layers as a nested hierarchical convolution of three motional processes: (i) individual motions described by the vibrational motions of H-atoms; (ii) internal motions including movements of the lipid backbone, head groups and tails, and (iii) molecule movements of the lipid molecule as a whole. The analytical expressions of the ISF associated with these movements are all derived. For use in analyzing the QENS experimental data, we also derived an analytical expression for the aggregate ISF of the Matryoshka model which involves an elastic term plus three inelastic terms of well-separated time scales and whose amplitudes and rates are functions of the lipid motions. And as an illustrative application, we used the aggregated ISF to analyze the experimental QENS data on a lipid sample of multilamellar bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). It is clear from this analysis that the dynamical Matryoshka model describes very well the experimental data and allow extracting the dynamical parameters of the studied system.
Assuntos
Dimiristoilfosfatidilcolina , Difração de Nêutrons , Membrana Celular , Bicamadas Lipídicas , NêutronsRESUMO
Biological membranes are generally formed by lipids and proteins. Often, the membrane properties are studied through model membranes formed by phospholipids only. They are molecules composed by a hydrophilic head group and hydrophobic tails, which can present a panoply of various motions, including small localized movements of a few atoms up to the diffusion of the whole lipid or collective motions of many of them. In the past, efforts were made to measure these motions experimentally by incoherent neutron scattering and to quantify them, but with upcoming modern neutron sources and instruments, such models can now be improved. In the present work, we expose a quantitative and exhaustive study of lipid dynamics on DMPC and DMPG membranes, using the Matryoshka model recently developed by our group. The model is confronted here to experimental data collected on two different membrane samples, at three temperatures and two instruments. Despite such complexity, the model describes reliably the data and permits to extract a series of parameters. The results compare also very well to other values found in the literature.
Assuntos
Difração de Nêutrons , Fosfolipídeos , Membrana Celular , Difusão , Membranas/química , Difração de Nêutrons/métodos , Fosfolipídeos/químicaRESUMO
NCYM, a cis-antisense gene of MYCN, encodes a Homininae-specific protein that promotes the aggressiveness of human tumors. Newly evolved genes from non-genic regions are known as de novo genes, and NCYM was the first de novo gene whose oncogenic functions were validated in vivo. Targeting NCYM using drugs is a potential strategy for cancer therapy; however, the NCYM structure must be determined before drug design. In this study, we employed vacuum-ultraviolet circular dichroism to evaluate the secondary structure of NCYM. The SUMO-tagged NCYM and the isolated SUMO tag in both hydrogenated and perdeuterated forms were synthesized and purified in a cell-free in vitro system, and vacuum-ultraviolet circular dichroism spectra were measured. Significant differences between the tagged NCYM and the isolated tag were evident in the wavelength range of 190-240 nm. The circular dichroism spectral data combined with a neural network system enabled to predict the secondary structure of NCYM at the amino acid level. The 129-residue tag consists of α-helices (approximately 14%) and ß-strands (approximately 29%), which corresponded to the values calculated from the atomic structure of the tag. The 238-residue tagged NCYM contained approximately 17% α-helices and 27% ß-strands. The location of the secondary structure predicted using the neural network revealed that these secondary structures were enriched in the Homininae-specific region of NCYM. Deuteration of NCYM altered the secondary structure at D90 from an α-helix to another structure other than α-helix and ß-strand although this change was within the experimental error range. All four nonsynonymous single-nucleotide polymorphisms (SNPs) in human populations were in this region, and the amino acid alteration in SNP N52S enhanced Myc-nick production. The D90N mutation in NCYM promoted NCYM-mediated MYCN stabilization. Our results reveal the secondary structure of NCYM and demonstrated that the Homininae-specific domain of NCYM is responsible for MYCN stabilization.
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The latest coronavirus SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19) pneumonia leading to the pandemic, contains 29 proteins. Among them, nucleocapsid protein (NCoV2) is one of the abundant proteins and shows multiple functions including packaging the RNA genome during the infection cycle. It has also emerged as a potential drug target. In this review, the current status of the research of NCoV2 is described in terms of molecular structure and dynamics. NCoV2 consists of two domains, i.e., the N-terminal domain (NTD) and the C-terminal domain (CTD) with a disordered region between them. Recent simulation studies have identified several potential drugs that can bind to NTD or CTD with high affinity. Moreover, it was shown that the degree of flexibility in the disordered region has a large effect on drug binding rate, suggesting the importance of molecular flexibility for the NCoV2 function. Molecular flexibility has also been shown to be integral to the formation of droplets, where NCoV2, RNA and/or other viral proteins gather through liquid-liquid phase separation and considered important for viral replication. Finally, as one of the future research directions, a strategy for obtaining the structural and dynamical information on the proteins contained in droplets is presented.
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To study the effects of the interdomain flexibility on the encounter rate of nucleocapsid-type protein with drug molecules, where two domains (NTD) are connected by a flexible linker and each NTD has a drug binding site, two-dimensional random walk simulation was carried out as a function of the interdomain flexibility and the drug concentration. NTDs represented as circles undergo random motions constrained by the interdomain flexibility while drug molecules are represented by lattice points. It was found that as the interdomain flexibility increases, the time interval between the drug bindings to the 1st and 2nd NTDs decreases, suggesting that the 2nd drug binding is accelerated. Furthermore, this effect was more significant at lower drug concentrations. These results suggest that the interdomain linker plays a key role in the drug binding process and thus emphasize the importance of characterization of their physicochemical properties to better evaluate the efficacy of potential drugs.
Assuntos
Antivirais/farmacologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , SARS-CoV-2/efeitos dos fármacos , Modelos Teóricos , Domínios Proteicos , SARS-CoV-2/metabolismoRESUMO
Lysozyme amyloidosis is a hereditary disease, which is characterized by the deposition of lysozyme amyloid fibrils in various internal organs. It is known that lysozyme fibrils show polymorphism and that polymorphs formed at near-neutral pH have the ability to promote more monomer binding than those formed at acidic pH, indicating that only specific polymorphs become dominant species in a given environment. This is likely due to the polymorph-specific configurational diffusion. Understanding the possible differences in dynamical behavior between the polymorphs is thus crucial to deepen our knowledge of amyloid polymorphism and eventually elucidate the molecular mechanism of lysozyme amyloidosis. In this study, molecular dynamics at sub-nanosecond timescale of two kinds of polymorphic fibrils of hen egg white lysozyme, which has long been used as a model of human lysozyme, formed at pH 2.7 (LP27) and pH 6.0 (LP60) was investigated using elastic incoherent neutron scattering (EINS) and quasi-elastic neutron scattering (QENS). Analysis of the EINS data showed that whereas the mean square displacement of atomic motions is similar for both LP27 and LP60, LP60 contains a larger fraction of atoms moving with larger amplitudes than LP27, indicating that the dynamical difference between the two polymorphs lies not in the averaged amplitude, but in the distribution of the amplitudes. Furthermore, analysis of the QENS data showed that the jump diffusion coefficient of atoms is larger for LP60, suggesting that the atoms of LP60 undergo faster diffusive motions than those of LP27. This study thus characterizes the dynamics of the two lysozyme polymorphs and reveals that the molecular dynamics of LP60 is enhanced compared with that of LP27. The higher molecular flexibility of the polymorph would permit to adjust its conformation more quickly than its counterpart, facilitating monomer binding.
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It is widely accepted that disordered regions in proteins, part of which takes predominantly α-helical conformation to a varying degree, play a critical role in biological function. Structural analysis of these flexible proteins is, however, not straightforward because existing methods to characterize the structural features of the disordered regions are not applicable to all the proteins with various sizes and physicochemical properties. In this study, to gain information on the helical propensity in the disordered regions as well as the overall three-dimensional structures of the proteins, usefulness of the combined small- and medium-angle X-ray scattering (SMAXS), which provides structural information at higher spatial resolution than commonly-used small-angle X-ray scattering, was investigated using computer simulations. For this purpose, various conformations of a protein that consists of two well-folded domains connected by a flexible linker were generated while the linker was modeled either to contain several α-helices or as random coils. The differences in the SMAXS scattering curves of these models were then evaluated. Analysis showed that the SMAXS curves allow to distinguish α-helices from random coils if at least â¼20% of all the residues in the proteins contributes to the helical formation in the disordered region, suggesting that structural analysis using SMAXS will be useful not only to obtain the three-dimensional domain organizations but also to gain information on the helical propensity in the disordered regions. This would lead to more accurate interpretation of reaction kinetics that the flexible proteins are involved in.
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Modelos Moleculares , Proteínas/química , Simulação por Computador , Conformação Proteica , Espalhamento de RadiaçãoRESUMO
Characterization of the dynamics of disordered polypeptide chains is required to elucidate the behavior of intrinsically disordered proteins and proteins under non-native states related to the folding process. Here we develop a method using quasielastic neutron scattering, combined with small-angle X-ray scattering and dynamic light scattering, to evaluate segmental motions of proteins as well as diffusion of the entire molecules and local side-chain motions. We apply this method to RNase A under the unfolded and molten-globule (MG) states. The diffusion coefficients arising from the segmental motions are evaluated and found to be different between the unfolded and MG states. The values obtained here are consistent with those obtained using the fluorescence-based techniques. These results demonstrate not only feasibility of this method but also usefulness to characterize the behavior of proteins under various disordered states.
Assuntos
Ribonuclease Pancreático/química , Espalhamento a Baixo Ângulo , Difusão , Difusão Dinâmica da Luz , Transferência de Energia , Espectroscopia de Ressonância Magnética , Difração de Nêutrons , Desdobramento de Proteína , Ribonuclease Pancreático/metabolismoRESUMO
α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of αSyn is sensitive to solution conditions, suggesting that fibril formation of αSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because intrinsically disordered proteins such as αSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering and small-angle x-ray scattering techniques, we investigated the dynamic and structural properties of αSyn under the conditions, where mature fibrils are formed (pHâ¯7.4 with a high salt concentration), where clumping of short fibrils occurs (pHâ¯4.0), and where fibril formation is not completed (pHâ¯7.4). The small-angle x-ray scattering measurements showed that the extended structures at pHâ¯7.4 with a high salt concentration become compact at pHâ¯4.0 and 7.4. The quasielastic neutron scattering measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pHâ¯7.4 with a high salt concentration, compared to those at pHâ¯7.4 without salt, whereas only the local motions are enhanced at pHâ¯4.0. These results imply that fibril formation of αSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.