RESUMEN
Medin amyloid, prevalent in the vessel walls of 97â¯% of individuals over 50, contributes to arterial stiffening and cerebrovascular dysfunction, yet our understanding of its aggregation mechanism remains limited. Dividing the full-length 50-amino-acid medin peptide into five 10-residue segments, we conducted individual investigations on each segment's self-assembly dynamics via microsecond-timescale atomistic discrete molecular dynamics (DMD) simulations. Our findings showed that medin1-10 and medin11-20 segments predominantly existed as isolated unstructured monomers, unable to form stable oligomers. Medin31-40 exhibited moderate aggregation, forming dynamic ß-sheet oligomers with frequent association and dissociation. Conversely, medin21-30 and medin41-50 segments demonstrated significant self-assembly capability, readily forming stable ß-sheet-rich oligomers. Residue pairwise contact frequency analysis highlighted the critical roles of residues 22-26 and 43-49 in driving the self-assembly of medin21-30 and medin41-50, acting as the ß-sheet core and facilitating ß-strand formation in other regions within medin monomers, expecting to extend to oligomers and fibrils. Regions containing residues 22-26 and 43-49, with substantial self-assembly abilities and assistance in ß-sheet formation, represent crucial targets for amyloid inhibitor drug design against aortic medial amyloidosis (AMA). In summary, our study not only offers deep insights into the mechanism of medin amyloid formation but also provides crucial theoretical and practical guidance for future treatments of AMA.
Asunto(s)
Amiloide , Simulación de Dinámica Molecular , Humanos , Amiloide/química , Amiloide/metabolismo , Aorta/metabolismo , Agregado de Proteínas , Péptidos/química , Péptidos/metabolismo , Conformación Proteica en Lámina beta , Antígenos de Superficie/metabolismo , Antígenos de Superficie/química , Secuencia de Aminoácidos , Proteínas de la LecheRESUMEN
A protein superfold is a type of protein fold that is observed in at least three distinct, non-homologous protein families. Structural classification studies have revealed a limited number of prevalent superfolds alongside several infrequent occurring folds, and in α/ß type superfolds, the C-terminal ß-strand tends to favor the edge of the ß-sheet, while the N-terminal ß-strand is often found in the middle. The reasons behind these observations, whether they are due to evolutionary sampling bias or physical interactions, remain unclear. This article offers a physics-based explanation for these observations, specifically for pure parallel ß-sheet topologies. Our investigation is grounded in several established structural rules that are based on physical interactions. We have identified "frustration-free topologies" which are topologies that can satisfy all the rules simultaneously. In contrast, topologies that cannot are termed "frustrated topologies." Our findings reveal that frustration-free topologies represent only a fraction of all theoretically possible patterns, these topologies strongly favor positioning the C-terminal ß-strand at the edge of the ß-sheet and the N-terminal ß-strand in the middle, and there is significant overlap between frustration-free topologies and superfolds. We also used a lattice protein model to thoroughly investigate sequence-structure relationships. Our results show that frustration-free structures are highly designable, while frustrated structures are poorly designable. These findings suggest that superfolds are highly designable due to their lack of frustration, and the preference for positioning C-terminal ß-strands at the edge of the ß-sheet is a direct result of frustration-free topologies. These insights not only enhance our understanding of sequence-structure relationships but also have significant implications for de novo protein design.
Asunto(s)
Modelos Moleculares , Pliegue de Proteína , Proteínas , Proteínas/química , Conformación Proteica en Lámina beta , Biología Computacional/métodosRESUMEN
Pathological aggregation of α-synuclein (α-syn) into amyloid fibrils is a major feature of Parkinson's disease (PD). The self-assembly of α-syn is mainly governed by a non-amyloid-ß component core (NACore). However, the effects of concentrations and temperatures on their conformational transition remain unclear. To answer this question, we investigated the aggregation kinetics of NACore oligomers in silico by performing several independent all-atom molecular dynamics simulations. The simulation results show that tetramers are more prone to form ß-sheets at 300 K than dimers and octamers. We also found that the NACore oligomers had higher ß-sheet and ß-barrel contents at 310 K. The inter-chain hydrophobic interactions, the backbone hydrogen bonding, the residue-residue interactions between V70-V77 as well as V77-V77 play important roles in the aggregation tendency of NACore octamers at 310 K. Interestingly, the energy gap analysis revealed that the conformational transition of NACore oligomers from intermediate states (ß-barrel conformation) to stable structures (ß-sheet layers) was dependent on the temperatures. In short, our study provides insight into the kinetic and thermodynamic mechanisms of the conformational transition of NACore at different concentrations and temperatures, contributing to a better understanding of the aggregation process of α-syn in Parkinson's disease.
Asunto(s)
Simulación de Dinámica Molecular , Temperatura , Termodinámica , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Conformación Proteica , Conformación Proteica en Lámina betaRESUMEN
Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a ß-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the ß1- and the ß12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the ß1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the ß1-strand with the ß3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the ß12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the ß-signals of classical autotransporter proteins and outer membrane ß-barrel proteins in general. We therefore propose that the ß12-strand of YeeJ acts as its ß-signal and that the assembly of the YeeJ ß-barrel is driven by its C-terminal ß-strand, like in most other autotransporter proteins, despite its inverted domain organization.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/química , Sistemas de Secreción Tipo V/metabolismo , Sistemas de Secreción Tipo V/genética , Sistemas de Secreción Tipo V/química , Modelos Moleculares , Mutación , Dominios Proteicos , Conformación Proteica en Lámina beta , Secuencia de AminoácidosRESUMEN
In this work, we elucidated the structural organization of stimuli-responsive peptide-polydiacetylene (PDA) conjugates that can self-assemble as 1D nanostructures under neutral aqueous conditions. The amino acid sequences bear positively or negatively charged domains at the periphery of the peptide segments to promote solubility in water while also driving assembly of the individual and combined components into ß-sheets. The photopolymerization of PDA, as well as the sensitivity of the resulting optical properties of the polymeric material to external stimuli, highly depends on the structural organization of the assembly of amphiphilic peptide-diacetylene units into 1D-nanostructures. Solid-state NMR measurements on 13C-labeled and 15N-labeled samples show that positively charged and negatively charged peptide amphiphiles are each capable of self-assembly, but self-assembly favors antiparallel ß-sheet structure. When positively and negatively charged peptide amphiphiles interact in stoichiometric solutions, cooperative coassembly dominates over self-assembly, resulting in the desired parallel ß-sheet structure with a concomitant increase in structural order. These results reveal that rational placement of oppositely charged residues can control ß-strand organization in a peptide amphiphile coassembly, which would have implications on the adaptive properties of stimuli-responsive biomaterials such as the peptide-PDAs studied here.
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Péptidos , Polímero Poliacetilénico , Péptidos/química , Polímero Poliacetilénico/química , Conformación Proteica en Lámina beta , Tensoactivos/química , Espectroscopía de Resonancia Magnética , Poliinos/química , Polímeros/química , Nanoestructuras/químicaRESUMEN
Beta turns, in which the protein backbone abruptly changes direction over four amino acid residues, are the most common type of protein secondary structure after alpha helices and beta sheets and play key structural and functional roles. Previous work has produced classification systems for turn geometry at multiple levels of precision, but these operate in backbone dihedral-angle (Ramachandran) space, and the absence of a local Euclidean-space coordinate system and structural alignment for turns, or of any systematic Euclidean-space characterization of turn backbone shape, presents challenges for the visualization, comparison and analysis of the wide range of turn conformations and the design of turns and the structures that incorporate them. This work derives a turn-local coordinate system that implicitly aligns turns, together with a set of geometric descriptors that characterize the bulk BB shapes of turns and describe modes of structural variation not explicitly captured by existing systems. These modes are shown to be meaningful by the demonstration of clear relationships between descriptor values and the electrostatic energy of the beta-turn H-bond, the overrepresentations of key side-chain motifs, and the structural contexts of turns. Geometric turn descriptors complement Ramachandran-space classifications, and they can be used to select turn structures for compatibility with particular side-chain interactions or contexts. Potential applications include protein design and other tasks in which an enhanced Euclidean-space characterization of turns may improve understanding or performance. The web-based tools ExploreTurns, MapTurns, and ProfileTurn, available at www.betaturn.com, incorporate turn-local coordinates and turn descriptors and demonstrate their utility.
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Modelos Moleculares , Proteínas , Proteínas/química , Enlace de Hidrógeno , Bases de Datos de Proteínas , Estructura Secundaria de Proteína , Electricidad Estática , Conformación Proteica en Lámina betaRESUMEN
Methods from artificial intelligence (AI), in general, and machine learning, in particular, have kept conquering new territories in numerous areas of science. Most of the applications of these techniques are restricted to the classification of large data sets, but new scientific knowledge can seldom be inferred from these tools. Here we show that an AI-based amyloidogenecity predictor can strongly differentiate the border- and the internal hexamers of ß-pleated sheets when screening all the Protein Data Bank-deposited homology-filtered protein structures. Our main result shows that more than 30% of internal hexamers of ß sheets are predicted to be amyloidogenic, while just outside the border regions, only 3% are predicted as such. This result may elucidate a general protection mechanism of proteins against turning into amyloids: if the borders of ß-sheets were amyloidogenic, then the whole ß sheet could turn more easily into an insoluble amyloid-structure, characterized by periodically repeated parallel ß-sheets. We also present that no analogous phenomenon exists on the borders of α-helices or randomly chosen subsequences of the studied protein structures.
Asunto(s)
Amiloide , Amiloide/química , Humanos , Conformación Proteica en Lámina beta , Bases de Datos de Proteínas , Modelos Moleculares , Inteligencia Artificial , Estructura Secundaria de ProteínaRESUMEN
Dynamic attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at both solutions and coatings of a semicrystalline silk material derived from Bombyx mori was applied to monitor the ß-sheet conformation, which is known to correlate with silk protein crystallinity. The secondary structure-sensitive Amide I band was analyzed. Two silk protein samples were studied: native-based silk buffer fibroin (NSF) was extracted from silk glands and regenerated silk fibroin (RSF) was extracted from degummed cocoons. Solutions of both NSF and RSF at 2 mg/mL featured low initial ß-sheet contents of 5-12%, which further increased to 47-53% after 24 h. RSF and NSF solutions at 23 mg/mL also featured low initial ß-sheet contents of 9-10%, which yet only slightly increased to 16-17% after 24 h. Coatings deposited from RSF solutions showed high surface integrity (Q > 99%) after rinsing in mineralized water, enabling interfacial drug delivery applications. RSF coatings were post-treated with either formic acid (FA) or pure methanol (MeOH) vapor to showcase inducibility of crystalline domains in RSF coatings. Such coatings were loaded with the model antibiotic drugs tetracycline (TCL) and streptomycin (STRP), and the sustained release of TCL was followed in contact with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer. RSF/TCL coatings post-treated with formic acid (FA) vapor followed by methanol (MeOH) vapor showed a significantly lower (52%) initial burst of rather hydrophobic TCL compared to untreated RSF/TCL coatings (72%), while no such significant release difference was observed for hydrophilic STRP. This was rationalized by a specific interaction between nonpolar TCL and hydrophobic crystalline RSF domains.
Asunto(s)
Bombyx , Fibroínas , Animales , Bombyx/química , Espectroscopía Infrarroja por Transformada de Fourier , Fibroínas/química , Conformación Proteica en Lámina beta , Liberación de Fármacos , Seda/química , Soluciones , Materiales Biocompatibles Revestidos/química , Tetraciclina/químicaRESUMEN
The secondary structures of polypeptides, such as an α-helix and a ß-sheet, often impart specific properties and functions, making the regulation of their secondary structures of great significance. Particularly, water-soluble polypeptides bearing a ß-sheet conformation are rare and challenging to achieve. Here, a series of oligo(ethylene glycol)-modified lysine N-carboxylic anhydrides (EGmK-NCA, where m = 1-3) and the corresponding polymers EGmKn are synthesized, with urethane bonds as the linker between the side-chain EG and lysine. The secondary structure of EGmKn is delicately regulated by both m and n, the length (number of repeating units) of EG and the degree of polymerization (DP), respectively. Among them, EG2Kn adopts a ß-sheet conformation with good water solubility at an appropriate DP and forms physically cross-linked hydrogels at a concentration as low as 1 wt %. The secondary structures of EG1Kn can be tuned by DP, exhibiting either a ß-sheet or an α-helix, whereas EG3Kn appears to a adopt pure and stable α-helix with no dependence on DP. Compared to previous works reporting EG-modified lysine-derived polypeptides bearing exclusively an α-helix conformation, this work highlights the important and unexpected role of the urethane connecting unit and provides useful case studies for understanding the secondary structure of polypeptides.
Asunto(s)
Péptidos , Conformación Proteica en Lámina beta , Solubilidad , Agua , Péptidos/química , Agua/química , Polietilenglicoles/química , Lisina/química , Hidrogeles/química , Glicol de Etileno/química , Estructura Secundaria de Proteína , PolimerizacionRESUMEN
The effect of ß-sheet ratio and chain length on all-ß proteins was investigated by MD simulations. Protein samples composed of different repeating units with various ß-sheet ratios or a different number of repeating units were simulated under a broad temperature range. The simulation results show that the smaller radius of gyration was achieved by the protein with the higher proportion of ß-sheet secondary structure, which had the lower nonbonded energy with more HBs within the protein. The root mean square deviation (RMSD) and the root mean square fluctuation (RMSF) both increased with temperature, especially in the case of a longer chain. The visible period was also shown according to the repeated secondary structure. Several minimum values of RMSF were located on the skeleton of Cα atoms participating in the ß-sheet, indicating that it is a kind of stable secondary structure. We also concluded that proteins with a short chain or a lower ratio of ß-sheet could easily transform their oriented and compact structures to other ones, such as random coils, turns, and even α-helices. These results clarified the relationship from the primary level to the 3D structure of proteins and potentially predicted protein folding.
Asunto(s)
Simulación de Dinámica Molecular , Conformación Proteica en Lámina beta , Proteínas , Proteínas/química , Pliegue de Proteína , Estructura Secundaria de Proteína , TemperaturaRESUMEN
Peptide self-assembly into amyloid fibrils provides numerous applications in drug delivery and biomedical engineering applications. We augment our previously-established computational screening technique along with experimental biophysical characterization to discover 7-mer peptides that self-assemble into "parallel ß-sheets", that is, ß-sheets with N-terminus-to-C-terminus ð½-strand vectors oriented in parallel. To accomplish the desired ß-strand organization, we applied the PepAD amino acid sequence design software to the Class-1 cross-ß spine defined by Sawaya et al. This molecular configuration includes two layers of parallel ß-sheets stacked such that N-terminus-to-C-terminus vectors are oriented antiparallel for molecules on adjacent ß-sheets. The first cohort of PepAD identified peptides were examined for their fibrillation behavior in DMD/PRIME20 simulations, and the top performing sequence was selected as a prototype for a subsequent round of sequence refinement. The two rounds of design resulted in a library of eight 7-mer peptides. In DMD/PRIME20 simulations, five of these peptides spontaneously formed fibril-like structures with a predominantly parallel ð½-sheet arrangement, two formed fibril-like structure with <50% in parallel ð½-sheet arrangement and one remained a random coil. Among the eight candidate peptides produced by PepAD and DMD/PRIME20, five were synthesized and purified. All five assembled into amyloid fibrils composed of parallel ß-sheets based on Fourier transform infrared spectroscopy, circular dichroism, electron microscopy, and thioflavin-T fluorescence spectroscopy measurements.
Asunto(s)
Método de Montecarlo , Conformación Proteica en Lámina beta , Nanofibras/química , Péptidos/química , Secuencia de Aminoácidos , Estructura Secundaria de Proteína , Amiloide/química , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
Although hydrophobic interactions provide the main driving force for initial peptide aggregation, their role in regulating suprastructure handedness of higher-order architectures remains largely unknown. We here interrogate the effects of hydrophobic amino acids on handedness at various assembly stages of peptide amphiphiles. Our studies reveal that relative to aliphatic side chains, aromatic side chains set the twisting directions of single ß-strands due to their strong steric repulsion to the backbone, and upon packing into multi-stranded ß-sheets, the side-chain aromatic interactions between strands form the aromatic ladders with a directional preference. This ordering not only leads to parallel ß-sheet arrangements but also induces the chiral flipping over of single ß-strands within a ß-sheet. In contrast, the lack of orientational hydrophobic interactions in the assembly of aliphatic peptides implies no chiral inversion upon packing into ß-sheets. This study opens an avenue to harness peptide aggregates with targeted handedness via aromatic side-chain interactions.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Péptidos , Péptidos/química , Péptidos/metabolismo , Conformación Proteica en Lámina beta , Estereoisomerismo , Estructura Secundaria de Proteína , Aminoácidos Aromáticos/química , Dicroismo Circular , Modelos Moleculares , Aminoácidos/químicaRESUMEN
Transmembrane ß-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane ß-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.
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Nanoporos , Ingeniería de Proteínas , Pliegue de Proteína , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Lámina beta , Modelos MolecularesRESUMEN
In a recent issue of Nature Chemical Biology, Emmanouilidis et al. (2024) investigate the maturation of biomolecular condensates of FUS1-267 and probe the molecular details of droplet aging. They observe that the liquid-to-solid transition of the droplet is mediated at the surface by FUS1-267 molecules that have adopted ß-strand conformations.
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Proteína FUS de Unión a ARN , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/química , Biofisica , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Humanos , Conformación Proteica en Lámina betaRESUMEN
Klebsiella pneumoniae (Kp) is an infectious disease pathogen that poses a significant global health threat due to its potential to cause severe infections and its tendency to exhibit multidrug resistance. Understanding the enzymatic mechanisms of the oxygen-insensitive nitroreductases (Kp-NRs) from Kp is crucial for the development of effective nitrofuran drugs, such as nitrofurantoin, that can be activated as antibiotics. In this paper, three crystal structures of two Kp-NRs (PDB entries 7tmf/7tmg and 8dor) are presented, and an analysis of their crystal structures and their flavin mononucleotide (FMN)-binding mode is provided. The structures with PDB codes 7tmf (Kp-NR1a), 7tmg (Kp-NR1b) and 8dor (Kp-NR2) were determined at resolutions of 1.97, 1.90 and 1.35â Å, respectively. The Kp-NR1a and Kp-NR1b structures adopt an αß fold, in which four-stranded antiparallel ß-sheets are surrounded by five helices. With domain swapping, the ß-sheet was expanded with a ß-strand from the other molecule of the dimer. The difference between the structures lies in the loop spanning Leu173-Ala185: in Kp-NR1a the loop is disordered, whereas the loop adopts multiple conformations in Kp-NR1b. The FMN interactions within Kp-NR1/NR2 involve hydrogen-bond and π-stacking interactions. Kp-NR2 contains four-stranded antiparallel ß-sheets surrounded by eight helices with two short helices and one ß-sheet. Structural and sequence alignments show that Kp-NR1a/b and Kp-NR2 are homologs of the Escherichia coli oxygen-insensitive NRs YdjA and NfnB and of Enterobacter cloacae NR, respectively. By homology inference from E. coli, Kp-NR1a/b and Kp-NR2 may detoxify polynitroaromatic compounds and Kp-NR2 may activate nitrofuran drugs to cause bactericidal activity through a ping-pong bi-bi mechanism, respectively.
Asunto(s)
Klebsiella pneumoniae , Modelos Moleculares , Nitrorreductasas , Klebsiella pneumoniae/enzimología , Cristalografía por Rayos X , Nitrorreductasas/química , Nitrorreductasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Secuencia de Aminoácidos , Mononucleótido de Flavina/metabolismo , Mononucleótido de Flavina/química , Sitios de Unión , Unión Proteica , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/enzimología , Conformación Proteica en Lámina beta , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
Affilin proteins, artificial binding proteins based on the ubiquitin scaffold, have been generated by directed protein evolution to yield de-novo variants that bind the extra-domain B (EDB) of oncofetal fibronectin, an established marker of tumor neovasculature. The crystal structures of two EDB-specific Affilin variants reveal a striking structural plasticity of the ubiquitin scaffold, characterised by ß-strand slippage, leading to different negative register shifts of the ß5 strands. This process recruits amino acid residues from ß5 towards the N-terminus to an adjacent loop region and subsequent residues into ß5, respectively, remodeling the binding interface and leading to target specificity and affinity. Protein backbone alterations resulting from ß-strand register shifts, as seen in the ubiquitin fold, can pose additional challenges to protein engineering as structural evidence of these events is still limited and they are difficult to predict. However, they can surface under the selection pressure of directed evolution and suggest that backbone plasticity allowing ß-strand slippages can increase structural diversity, enhancing the evolutionary potential of a protein scaffold.
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Fibronectinas , Ubiquitina , Fibronectinas/metabolismo , Fibronectinas/química , Fibronectinas/genética , Ubiquitina/metabolismo , Humanos , Unión Proteica , Conformación Proteica en Lámina beta , Modelos Moleculares , Cristalografía por Rayos X , Ingeniería de ProteínasRESUMEN
Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-ß (Aß) peptides of varying lengths. Most cleavages have a step size of three residues. To elucidate the underlying mechanism, we determined the atomic structures of human γ-secretase bound individually to APP-C99, Aß49, Aß46, and Aß43. In all cases, the substrate displays the same structural features: a transmembrane α-helix, a three-residue linker, and a ß-strand that forms a hybrid ß-sheet with presenilin 1 (PS1). Proteolytic cleavage occurs just ahead of the substrate ß-strand. Each cleavage is followed by unwinding and translocation of the substrate α-helix by one turn and the formation of a new ß-strand. This mechanism is consistent with existing biochemical data and may explain the cleavages of other substrates by γ-secretase.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Presenilina-1 , Humanos , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/química , Cristalografía por Rayos X , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/química , Presenilina-1/química , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteolisis , Especificidad por SustratoRESUMEN
The effect of halide substitution in Grubbs-Hoveyda II catalysts (GHII catalysts) embedded in the engineered ß-barrel protein nitrobindin (NB4exp) on metathesis activity in aqueous media was studied. Maleimide tagged dibromido and diiodido derivates of the GHII catalyst were synthesized and covalently conjugated to NB4exp. The biohybrid catalysts were characterized spectroscopically confirming the structural integrity. When the two chloride substituents at ruthenium center were exchanged against bromide and iodide, the diiodo derivative was found to show significantly higher catalytic activity in ring-closing metathesis of α,ω-diolefins, whereas the dibromido derivative was less efficient when compared with the parent dichlorido catalyst. Using the diiodido catalyst, high turnover numbers of up to 75 were observed for ring-closing metathesis (RCM) yielding unsaturated six- and seven-membered N-heterocycles.
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Alquenos , Catálisis , Alquenos/química , Rutenio/química , Conformación Proteica en Lámina betaRESUMEN
The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant Plumbago zeylanica, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant Ka â¼ 10-4 M-1)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of ß-sheet (â¼18%) and disordered (â¼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native ß-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.
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Amiloide , Naftoquinonas , Agregado de Proteínas , Naftoquinonas/química , Naftoquinonas/metabolismo , Humanos , Amiloide/química , Amiloide/metabolismo , Cinética , Conformación Proteica en Lámina beta , Unión Proteica , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo , Albúmina Sérica Humana/genética , Albúmina Sérica/química , Albúmina Sérica/metabolismoRESUMEN
Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using in silico tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure. Introduction of proline residues into ß-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic to cells overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in ß-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that ß-strands II and III may be good targets for the development of SOD1-associated ALS therapies.