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Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.
Assuntos
Amiloide , Insulina , Amiloide/química , Proteínas Amiloidogênicas/metabolismo , Insulina/metabolismo , Ligação ProteicaRESUMO
HspB1 is a mammalian sHsp that is ubiquitously expressed in almost all tissues and involved in regulating many vital functions. Although the recent crystal structure of human HspB1 showed that 24 monomers form the oligomeric complex of human HspB1 in a spherical configuration, the molecular architecture of HspB1 is still controversial. In this study, we examined the oligomeric structural change of CgHspB1 by sedimentation velocity analytical ultracentrifugation. At the low temperature of 4 °C, CgHspB1 exists as an 18-mer, probably a trimeric complex of hexamers. It is relatively unstable and partially dissociates into small oligomers, hexamers, and dodecamers. At elevated temperatures, the 24-mer was more stable than the 18-mer. The 24-mer is also in dynamic equilibrium with the dissociated oligomers in the hexameric unit. The hexamer further dissociates to dimers. The disulfide bond between conserved cysteine residues seems to be partly responsible for the stabilization of hexamers. The N-terminal domain is involved in the assembly of dimers and the interaction between hexamers. It is plausible that CgHspB1 expresses a chaperone function in the 24-mer structure.
Assuntos
Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Conformação Proteica , Multimerização Proteica , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Domínios ProteicosRESUMO
The self-assembly of organic molecules into supramolecular materials with structural ordering beyond the nanometre scale is challenging. Here, we report the spontaneous self-assembly of a chiral discotic triphenylene derivative into millimetre-sized droplets. The structure of the droplets is characterized by high positional and orientational ordering and a three-dimensional integrity similar to that of single crystals. Notwithstanding, these assemblies slide when placed on a vertical substrate demonstrating their fluid nature. X-ray imaging shows that during the sliding process the internal crystal-like structure is maintained and that the droplets undergo clockwise or counterclockwise unidirectional rotation, depending on the chirality of their molecular components. Rheological measurements suggest that this rotational behaviour might result from the distinct yield stress between the (R)- and (S)-enantiomers. Overall, our findings demonstrate that molecular chirality can determine the movement direction of a supramolecular structure, thus expanding the fundamental understanding of the structure and dynamics of soft materials.
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Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.
Assuntos
Proteínas de Choque Térmico/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , HumanosRESUMO
Tetra-poly(acrylic acid) (tetra-PAA) gels were fabricated by cross-end coupling of azide (-N3) and alkyne (-C≡CH) terminated tetra-arm poly(tert-butyl acrylate)s. The structures of the tetra-PAA gels and their sols (tetra-PAA sols, i.e., tetra-PAA macromer solutions obtained by deprotection of tert-butyl groups) were investigated by small-angle X-ray and small-angle neutron scattering experiments in methanol, salt-free aqueous media, and salt solutions. The scattering functions, I(q), of the tetra-PAA sols in methanol were described by the Ornstein-Zernike (OZ) function, I q = I ( 0 ) / ( 1 + ξ 2 q 2 ) , whereas those of the gels were represented by the sum of the OZ function and a power-law function, I q â¼ A q b . Here, ξ is the correlation length, q is the magnitude of the scattering vector, A is a constant, and b is the scattering exponent. The OZ and the power-law functions represent the concentration fluctuations and cross-link inhomogeneities characteristic of gels, respectively. In salt-free aqueous media, there appeared a characteristic polyelectrolyte peak in the scattering functions, and the polymer concentration, C, dependence of the peak was similar for both sols and gels. In the presence of salt, both the scattering upturn for gels and the polyelectrolyte peak for both gels and sols were suppressed. Upon increasing the salt concentration, C s, the scattering intensity increased in the low q regions and the scattering functions were well fitted by the OZ function for both gels and sols.
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The structure and rheological properties of wormlike micelles formed by a fluorocarbon-hydrocarbon-type hybrid gemini surfactant in an aqueous solution were investigated by means of small-angle X-ray scattering (SAXS) and viscoelastic measurements. The cross-sectional structure (the radius of the hydrophobic core and the thickness of the hydrophilic shell) and the aggregation number per unit axial length of wormlike micelles were evaluated by a model fitting analysis of SAXS profiles. Both parameters for the hybrid gemini surfactant were smaller than those of a corresponding hydrocarbon-hydrocarbon-type gemini surfactant. On the other hand, the viscosity of the hybrid gemini surfactant was higher than that of the hydrocarbon-hydrocarbon-type gemini surfactant. From the viscoelastic parameters, the steady state compliance, Je, and the terminal relaxation time, τw, which were independently obtained by dynamic viscoelastic measurement, we revealed that a larger number of entanglements and a longer contour length of the hybrid gemini surfactant led to the higher viscosity. These results obtained by the rheological measurements were consistent with those obtained by SAXS analysis.
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We investigated the structure of the hydrophobe-uptake micelle of an alternating amphiphilic copolymer in aqueous solutions, by combining light scattering and small-angle X-ray scattering (SAXS). When the copolymer micelle includes the hydrophobe (1-dodecanol), the unicore flower micelle transforms into the multicore flower necklace, and the flower necklace is slightly stiffer than the hydrophobe-free flower necklace of the same copolymer. Moreover, the hydrophobe is included not in the hydrophobic core region but in the intermingled region of the hydrophobic group and the loop chain of the unit flower micelle. Therefore, the structure of the hydrophobe-uptake micelle of the amphiphilic alternating copolymer is quite different from that of hydrophobe-uptake spherical micelles of low molar mass surfactants and of amphiphilic block copolymers, where the hydrophobe is included in the hydrophobic region of the micelles.
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We have investigated aqueous micellar solutions of mixtures of a surfactant (dodecylpyridinium chloride) and a hydrophobe (1-dodecanol) near the critical micelle concentration (cmc), using simultaneous static and dynamic light scattering measurements. The static light scattering intensity for the aqueous solutions was separated into fast and slow relaxation components using dynamic light scattering results. The slow relaxation component gave us the information about the large scattering component. It turned out from this component that the amount of large colloidal particles of the hydrophobe was very tiny in the solution and hardly affects the association-dissociation equilibrium of the hydrophobe-uptake micelle. The free surfactant molecule and the hydrophobe-uptake spherical micelle in the solutions belong to the fast relaxation component. We have characterized the spherical micelle and also analyzed the association-dissociation equilibrium of the hydrophobe-uptake micelle up to near the cmc, using this scattering component extracted.
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Conserved tryptophan residues are critical for the structure and the stability of ß/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in ß/γ-crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of ß/γ-crystallin, which correlates with age-related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human ßB2-crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo-oligomerization of ßB2-crystallin-W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of ßB2-crystallin vary over time. By using SAXS, we found that the dimer of ßB2-crystallin in solution resembled the lattice ßB1-crystallin dimer (face-en-face), whereas the tetramer of ßB2-crystallin in solution resembled its lattice tetramer (domain-swapped). Our results suggest that homo-oligomerization of ßB2-crystallin includes potential inter-subunit reactions, such as dissociation, unfolding, and re-formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of ßB2-crystallin in lens.
Assuntos
Dobramento de Proteína , Triptofano , Cadeia B de beta-Cristalina , Humanos , Triptofano/química , Triptofano/genética , Cadeia B de beta-Cristalina/química , Cadeia B de beta-Cristalina/genética , Cadeia B de beta-Cristalina/metabolismo , Mutação , Multimerização Proteica , Estabilidade Proteica , Interações Hidrofóbicas e Hidrofílicas , Substituição de AminoácidosRESUMO
The nucleosome is a fundamental unit of chromatin in which about 150 base pairs of DNA are wrapped around a histone octamer. The overlapping di-nucleosome has been proposed as a product of chromatin remodeling around the transcription start site, and previously found as a chromatin unit, in which about 250 base pairs of DNA continuously bind to the histone core composed of a hexamer and an octamer. In the present study, our genome-wide analysis of human cells suggests another higher nucleosome stacking structure, the overlapping tri-nucleosome, which wraps about 300-350 base-pairs of DNA in the region downstream of certain transcription start sites of actively transcribed genes. We determine the cryo-electron microscopy (cryo-EM) structure of the overlapping tri-nucleosome, in which three subnucleosome moieties, hexasome, hexasome, and octasome, are associated by short connecting DNA segments. Small angle X-ray scattering and coarse-grained molecular dynamics simulation analyses reveal that the cryo-EM structure of the overlapping tri-nucleosome may reflect its structure in solution. Our findings suggest that nucleosome stacking structures composed of hexasome and octasome moieties may be formed by nucleosome remodeling factors around transcription start sites for gene regulation.
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Histonas , Nucleossomos , Humanos , Nucleossomos/genética , Microscopia Crioeletrônica , Histonas/genética , Cromatina , DNA/genéticaRESUMO
Early phase of amyloid formation, where prefibrillar aggregates such as oligomers and protofibrils are often observed, is crucial for understanding pathogenesis. However, the detailed mechanisms of their formation have been difficult to elucidate because they tend to form transiently and heterogeneously. Here, we found that bovine insulin protofibril formation proceeds in a monodisperse manner, which allowed us to characterize the detailed early aggregation process by light scattering in combination with thioflavin T fluorescence and Fourier transform infrared spectroscopy. The protofibril formation was specific to bovine insulin, whereas no significant aggregation was observed in human insulin. The kinetic analysis combining static and dynamic light scattering data revealed that the protofibril formation process in bovine insulin can be divided into two steps based on fractal dimension. When modeling the experimental data based on Smoluchowski aggregation kinetics, an aggregation scheme consisting of initial fractal aggregation forming spherical oligomers and their subsequent end-to-end association forming protofibrils was clarified. Furthermore, the analysis of temperature and salt concentration dependencies showed that the end-to-end association is the rate-limiting step, involving dehydration. The established model for protofibril formation, wherein oligomers are incorporated as a precursor, provides insight into the molecular mechanism by which protein molecules assemble during the early stage of amyloid formation.
Assuntos
Amiloide , Insulinas , Animais , Bovinos , Humanos , Amiloide/química , Insulinas/química , Cinética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Aggregates cause a fatal problem in the structural analysis of a biomacro-mol-ecule in solution using small-angle X-ray or neutron scattering (SAS): they deteriorate the scattering profile of the target molecule and lead to an incorrect structure. Recently, an integrated method of analytical ultracentrifugation (AUC) and SAS, abbreviated AUC-SAS, was developed as a new approach to overcome this problem. However, the original version of AUC-SAS does not offer a correct scattering profile of the target molecule when the weight fraction of aggregates is higher than ca 10%. In this study, the obstacle point in the original AUC-SAS approach is identified. The improved AUC-SAS method is then applicable to a solution with a relatively larger weight fraction of aggregates (≤20%).
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Pioneer factors, which can directly bind to nucleosomes, have been considered to change chromatin conformations. However, the binding impact on the nucleosome is little known. Here, we show how the pioneer factor GATA3 binds to nucleosomal DNA and affects the conformation and dynamics of nucleosomes by using a combination of SAXS, molecular modeling, and molecular dynamics simulations. Our structural models, consistent with the SAXS data, indicate that only one of the two DNA binding domains, N- and C-fingers, of GATA3 binds to an end of the DNA in solution. Our MD simulations further showed that the other unbound end of the DNA increases the fluctuation and enhances the DNA dissociation from the histone core when the N-finger binds to a DNA end, a site near the entry or exit of the nucleosome. However, this was not true for the binding of the C-finger that binds to a location about 15 base pairs distant from the DNA end. In this case, DNA dissociation occurred on the bound end. Taken together, we suggest that the N-finger and C-finger bindings of GATA3 commonly enhance DNA dissociation at one of the two DNA ends (the bound end for the C-finger binding and the unbound end for the N-finger binding), leading to triggering a conformational change in the chromatin.
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Fator de Transcrição GATA3 , Nucleossomos , Cromatina/química , DNA/química , Simulação de Dinâmica Molecular , Nucleossomos/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Ligação Proteica , Fator de Transcrição GATA3/química , Domínios ProteicosRESUMO
Vitamin Ks are expected to contribute bone and cardiovascular health. Especially, menaquinone-7 has a higher bioavailability and a longer half-life than other vitamin Ks in the human body. However, their low water-solubility limits their application. On the other hand, Bacillus subtilis natto produces a water-soluble complex, which comprises menaquinone-7 and peptides. The peptide named K-binding factor (KBF) has been reported as the main component of the complex. In the present, the structural characteristics of KBF were studied. Mass spectrometry showed significant peaks at m/z = 1050, while the previous PAGE suggested that molecular weight of KBF was ~ 3k. Amino acid analysis revealed that the 1k peptides were the various combinations of nine amino acids, among which Asx, Glx, Val, Leu and Met were found to be the most abundant. The peptides could serve as detergent properties. The 1k peptides could be isolated by reverse-phase high performance liquid chromatography. The bundle of three 1k detergent-like peptides would participate to the micelle structure containing menqauinone-7 inside. In conclusion, a basic unit of KBF would be the ~ 1k peptides, and the three basic unit assemble to the ~ 3k bundle, then the bundle form a water-soluble micelle including menqauinone-7 inside.
Assuntos
Bacillus subtilis , Alimentos de Soja , Humanos , Bacillus subtilis/metabolismo , Detergentes/metabolismo , Micelas , Vitamina K 2/metabolismo , Aminoácidos/metabolismo , Vitaminas/metabolismoRESUMO
Solving structural ensembles of flexible biomolecules is a challenging research area. Here, we propose a method to obtain possible structural ensembles of a biomolecule based on small-angle X-ray scattering (SAXS) and molecular dynamics simulations. Our idea is to clip a time series that matches a SAXS profile from a simulation trajectory. To examine its practicability, we applied our idea to a multi-domain protein ER-60 and successfully extracted time series longer than 1 micro second from trajectories of coarse-grained molecular dynamics simulations. In the extracted time series, the domain conformation was distributed continuously and smoothly in a conformational space. Preferred domain conformations were also observed. Diversity among scattering curves calculated from each ER-60 structure was interpreted to reflect an open-close motion of the protein. Although our approach did not provide a unique solution for the structural ensemble of the biomolecule, each extracted time series can be an element of the real behavior of ER-60. Considering its low computational cost, our approach will play a key role to identify biomolecular dynamics by integrating SAXS, simulations, and other experiments.
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Simulação de Dinâmica Molecular , Proteínas , Conformação Proteica , Proteínas/química , Espalhamento a Baixo Ângulo , Fatores de Tempo , Difração de Raios X , Raios XRESUMO
In the cyanobacterial circadian clock system, KaiA, KaiB and KaiC periodically assemble into a large complex. Here we determined the overall structure of their fully assembled complex by integrating experimental and computational approaches. Small-angle X-ray and inverse contrast matching small-angle neutron scatterings coupled with size-exclusion chromatography provided constraints to highlight the spatial arrangements of the N-terminal domains of KaiA, which were not resolved in the previous structural analyses. Computationally built 20 million structural models of the complex were screened out utilizing the constrains and then subjected to molecular dynamics simulations to examine their stabilities. The final model suggests that, despite large fluctuation of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions. Thus, our integrative approach provides a useful tool to resolve large complex structures harboring dynamically fluctuating domains.
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Relógios Circadianos , Cianobactérias , Proteínas de Bactérias/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Espalhamento a Baixo ÂnguloRESUMO
Multi-domain proteins (MDPs) show a variety of domain conformations under physiological conditions, regulating their functions through such conformational changes. One of the typical MDPs, ER-60 which is a protein folding enzyme, has a U-shape with four domains and is thought to have different domain conformations in solution depending on the redox state at the active centres of the edge domains. In this work, an aggregation-free small-angle X-ray scattering revealed that the structures of oxidized and reduced ER-60 in solution are different from each other and are also different from those in the crystal. Furthermore, structural modelling with coarse-grained molecular dynamics simulation indicated that the distance between the two edge domains of oxidized ER-60 is longer than that of reduced ER-60. In addition, one of the edge domains has a more flexible conformation than the other.
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Simulação de Dinâmica Molecular , Agregados Proteicos , Isomerases de Dissulfetos de Proteínas/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Humanos , Oxirredução , Domínios Proteicos , SoluçõesRESUMO
H2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate "open conformation", in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome.
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Montagem e Desmontagem da Cromatina , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Sítios de Ligação , Humanos , Microscopia de Força Atômica , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade , Fatores de TempoRESUMO
AlphaB crystallin (αB-crystallin) is a key protein for maintaining the long-term transparency of the eye lens. In the eye lens, αB-crystallin is a "dynamical" oligomer regulated by subunit exchange between the oligomers. To elucidate the unsettled mechanism of subunit exchange in αB-crystallin oligomers, the study was carried out at two different protein concentrations, 28.5 mg/mL (dense sample) and 0.45 mg/mL (dilute sample), through inverse contrast matching small-angle neutron scattering. Interestingly, the exchange rate of the dense sample was the same as that of the dilute sample. From analytical ultracentrifuge measurements, the coexistence of small molecular weight components and oligomers was detected, regardless of the protein concentration. The model proposed that subunit exchange could proceed through the assistance of monomers and other small oligomers; the key mechanism is attaching/detaching monomers and other small oligomers to/from oligomers. Moreover, this model successfully reproduced the experimental results for both dense and dilute solutions. It is concluded that the monomer and other small oligomers attaching/detaching mainly regulates the subunit exchange in αB-crystallin oligomer.
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The distinguished feature of neutron as a scattering probe is an isotope effect, especially the large difference in neutron scattering length between hydrogen and deuterium. The difference renders the different visibility between hydrogenated and deuterated proteins. Therefore, the combination of deuterated protein and neutron scattering enables the selective visualization of a target domain in the complex or a target protein in the multi-component system. Despite of this fascinating character, there exist several problems for the general use of this method: difficulty and high cost for protein deuteration, and control and determination of deuteration ratio of the sample. To resolve them, the protocol of protein deuteration techniques is presented in this report. It is strongly expected that this protocol will offer more opportunity for conducting the neutron scattering studies with deuterated proteins.