共查询到20条相似文献,搜索用时 15 毫秒
1.
Karen E. Marshall Thomas L. Williams Alison Rodger Louise C. Serpell 《Biophysical journal》2010,98(2):330-338
Amyloid-like fibrils can be formed by many different proteins and peptides. The structural characteristics of these fibers are very similar to those of amyloid fibrils that are deposited in a number of protein misfolding diseases, including Alzheimer's disease and the transmissible spongiform encephalopathies. The elucidation of two crystal structures from an amyloid-like fibril-forming fragment of the yeast prion, Sup35, with sequence GNNQQNY, has contributed to knowledge regarding side-chain packing of amyloid-forming peptides. Both structures share a cross-β steric zipper arrangement but vary in the packing of the peptide, particularly in terms of the tyrosine residue. We investigated the fibrillar and crystalline structure and assembly of the GNNQQNY peptide using x-ray fiber diffraction, electron microscopy, intrinsic and quenched tyrosine fluorescence, and linear dichroism. Electron micrographs reveal that at concentrations between 0.5 and 10 mg/mL, fibers form initially, followed by crystals. Fluorescence studies suggest that the environment of the tyrosine residue changes as crystals form. This is corroborated by linear dichroism experiments that indicate a change in the orientation of the tyrosine residue over time, which suggests that a structural rearrangement occurs as the crystals form. Experimental x-ray diffraction patterns from fibers and crystals also suggest that these species are structurally distinct. A comparison of experimental and calculated diffraction patterns contributes to an understanding of the different arrangements accessed by the peptide. 相似文献
2.
Amyloidlike fibrils are found in many fatal diseases, including Alzheimer's disease, type II diabetes mellitus, transmissible spongiform encephalopathies, and prion diseases. These diseases are linked to proteins that have partially unfolded, misfolded, and aggregated into amyloidlike fibrils. The kinetics of amyloidlike fibrils aggregation is still hotly debated and remains an important open question. We have utilized the GNNQQNY crystal structure and high-temperature molecular dynamics simulation in explicit solvent to study the disaggregation mechanism of the GNNQQNY fibrils and to infer its likely aggregation pathways. A hexamer model and a 12-mer model both with two parallel β-sheets separated by a dry side-chain interface were adopted in our computational analysis. A cumulative time of 1 μs was simulated for the hexamer model at five different temperatures (298 K, 348 K, 398 K, 448 K, and 498 K), and a cumulative time of 2.1 μs was simulated for the 12-mer model at four temperatures (298 K, 398 K, 448 K, and 498 K). Our disaggregation landscape and kinetics analyses indicate that tetramers probably act as the transition state in both the hexamer and the 12-mer simulations. In addition, the 12-mer simulations show that the initial aggregation nucleus is with eight peptides. Furthermore, the landscape is rather flat from 8-mers to 12-mers, indicating the absence of major barriers once the initial aggregation nucleus forms. Thus, the likely aggregation pathway is from monomers to the initial nucleus of 8-mers with tetramers as the transition state. Transition state structure analysis shows that the two dominant transition state conformations are tetramers in the 3-1 and 2-2 arrangements. The predominant nucleus conformations are in peptide arrangements maximizing dry side-chain contacts. Landscape and kinetics analyses also indicate that the parallel β-sheets form earlier than the dry side-chain contacts during aggregation. These results provide further insights in understanding the early fibrils aggregation. 相似文献
3.
Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutzfeldt‐Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full‐length amyloid proteins is not necessary for understanding amyloid formation. In this study, we simulate GNNQQNY, the N‐terminal prion‐determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. Utilizing a coarse‐grained model permits equilibration on relevant time scales. Replica‐exchange molecular dynamics is used to gather simulation statistics at multiple temperatures and clear energy traps that would aversely impact results. Investigating the association of 3‐, 6‐, and 12‐chain GNNQQNY systems by calculating thermodynamic quantities and orientational order parameters, we determine the aggregation pathway by studying aggregation states of GNNQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H‐bond formation, leading to the formation of β‐sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak. Proteins 2013; 81:1141–1155. © 2013 Wiley Periodicals, Inc. 相似文献
4.
《Journal of molecular biology》2021,433(3):166732
An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T fluorescence, Congo-red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases. 相似文献
5.
As research progresses toward understanding the role of the amyloid‐β (Aβ) peptide in Alzheimer's disease, certain aspects of the aggregation process for Aβ are still not clear. In particular, the accepted constitution of toxic aggregates in neurons has shifted toward small oligomers. However, the process of forming these oligomers in cells is also not full clear. Even more interestingly, it has been implied that cell membranes, and, in particular, anionic lipids within those membranes, play a key role in the progression of Aβ aggregation, but the exact nature of the Aβ‐membrane interaction in this process is unknown. In this work, we use a thermodynamic cycle and umbrella sampling molecular dynamics to investigate dimerization of the 42‐residue Aβ peptide on model zwitterionic dipalmitoylphosphatidylcholine (DPPC) or model anionic dioleoylphosphatidylserine (DOPS) bilayer surfaces. We determined that Aβ dimerization was strongly favored through interactions with the DOPS bilayer. Further, our calculations showed that the DOPS bilayer promoted strong protein–protein interactions within the Aβ dimer, whereas DPPC favored strong protein–lipid interactions. By promoting dimer formation and subsequent dimer release into the solvent, the DOPS bilayer acts as a catalyst in Aβ aggregation. Proteins 2010. © 2010 Wiley‐Liss, Inc. 相似文献
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Vitagliano L Esposito L Pedone C De Simone A 《Biochemical and biophysical research communications》2008,377(4):1036-1041
Protein and peptide aggregation into amyloid plaques is associated with a large variety of neurodegenerative diseases. The definition of the molecular bases of these pathologies is hampered by the transient nature of pre-fibrillar small-oligomers that are considered the toxic species. The ability of the peptide GNNQQNY to form amyloid-like structures makes it a good model to investigate the complex processes involved into amyloid fiber formation. By employing full atomistic replica exchange molecular dynamics simulations, we constructed the free energy surface of small assemblies of GNNQQNY to gain novel insights into the fiber formation process. The calculations suggest that the peptide exhibits a remarkable tendency to form both parallel and antiparallel β-sheets. The data show that GNNQQNY preference for parallel or antiparallel β-sheets is governed by a subtle balance of factors including assemblies’ size, sidechain-sidechain interactions and pH. The samplings analysis provides a rationale to the observed trends. 相似文献
8.
Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)10, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012. 相似文献
9.
A variational model for oligomer-formation process of GNNQQNY peptide from yeast prion protein Sup35
Many human neurodegenerative diseases are associated with the aggregation of insoluble amyloid-like fibrous proteins. However, the processes by which the randomly diffused monomer peptides aggregate into the highly regulated amyloid fibril structures are largely unknown. We proposed a residue-level coarse-grained variational model for the investigation of the aggregation pathway for a small assembly of amyloid proteins, the peptide GNNQQNY from yeast prion protein Sup35. By examining the free energy surface, we identified the residue-level sequential pathways for double parallel and antiparallel β-peptides, which show that the central dry polar zipper structure is the major folding core in both cases. The critical nucleus size is determined to be three peptides for the homogeneous nucleation process, whereas the zig-zag growth pattern appears most favorably for heterogeneous nucleation. Consistent with the dock-and-lock mechanism, the aggregation process of free peptides to the fibril core was found to be highly cooperative. The quantitative validation with the computational simulations and experimental data demonstrated the usefulness of the proposed model in understanding the general mechanism of the amyloid fibril system. 相似文献
10.
Jiho Kim ) Honggun Lee ) Joo Hyun Lee ) Do-yoon Kwon ) Auguste Genovesio Denis Fenistein Arnaud Ogier Vincent Brondani Regis Grailhe 《The Journal of biological chemistry》2014,289(21):15094-15103
More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins. 相似文献
11.
The early stages of peptide aggregation are currently not accessible by experimental techniques at atomic resolution. In this article, we address this problem through the application of a mixed simulation scheme in which a preliminary coarse-grained Monte Carlo analysis of the free-energy landscape is used to identify representative conformations of the aggregates and subsequent all-atom molecular dynamics simulations are used to analyze in detail possible pathways for the stabilization of oligomers. This protocol was applied to systems consisting of multiple copies of the model peptide GNNQQNY, whose detailed structures in the aggregated state have been recently solved in another study. The analysis of the various trajectories provides dynamical and structural insight into the details of aggregation. In particular, the simulations suggest a hierarchical mechanism characterized by the initial formation of stable parallel β-sheet dimers and identify the formation of the polar zipper motif as a fundamental feature for the stabilization of initial oligomers. Simulation results are consistent with experimentally derived observations and provide an atomically detailed view of the putative initial stages of fibril formation. 相似文献
12.
Lida Xu Ting-Ting Hu Shi-Zhong Luo 《International journal of peptide research and therapeutics》2014,20(1):95-102
E-cadherin is a transmembrane glycoprotein which is involved in the Ca2+-dependent cell–cell adhesion, and the adhesiveness is heavily dependent on the homodimerization of this molecule. Previous studies have shown that both the extracellular domain and cytoplasmic domain of E-cadherin contribute to its homodimerization. However, the roles of the transmembrane(TM) domain in the E-cadherin homodimerization have not been discussed in detail. In our experiments, SDS-PAGE showed higher molecular weight bands for the synthetic E-cadherin TM peptide, which indicated that the E-cadherin TM peptide is able to dimerize in the SDS micelle. The TOXCAT assay proved that the E-cadherin TM domain can form a moderate homo-oligomer in the Escherichia coli inner membrane. Furthermore, mutational analyses using the TOXCAT assays revealed that, instead of the common GxxxG dimerization motif, the leucine zipper motif is essential for the dimerization of the E-cadherin TM domain. Combining our experiment data and the computational simulation results, we provide insights for understanding the roles of the TM domain in the E-cadherin dimerization. 相似文献
13.
《Journal of molecular biology》2021,433(20):167160
Protein design has come of age, but how will it mature? In the 1980s and the 1990s, the primary motivation for de novo protein design was to test our understanding of the informational aspect of the protein-folding problem; i.e., how does protein sequence determine protein structure and function? This necessitated minimal and rational design approaches whereby the placement of each residue in a design was reasoned using chemical principles and/or biochemical knowledge. At that time, though with some notable exceptions, the use of computers to aid design was not widespread. Over the past two decades, the tables have turned and computational protein design is firmly established. Here, I illustrate this progress through a timeline of de novo protein structures that have been solved to atomic resolution and deposited in the Protein Data Bank. From this, it is clear that the impact of rational and computational design has been considerable: More-complex and more-sophisticated designs are being targeted with many being resolved to atomic resolution. Furthermore, our ability to generate and manipulate synthetic proteins has advanced to a point where they are providing realistic alternatives to natural protein functions for applications both in vitro and in cells. Also, and increasingly, computational protein design is becoming accessible to non-specialists. This all begs the questions: Is there still a place for minimal and rational design approaches? And, what challenges lie ahead for the burgeoning field of de novo protein design as a whole? 相似文献
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Fushinobu S Mertz B Hill AD Hidaka M Kitaoka M Reilly PJ 《Carbohydrate research》2008,343(6):1023-1033
GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into α-d-glucose 1-phosphate (G1P) and d-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites −1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. autodock was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy 4C1 conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from 1S3 as the pre-transition state conformer (Michaelis complex) via E3 as the transition state candidate to 4C1 as the G1P product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The 1S3 pre-transition state conformer is highly stable compared with other conformers, and a conformational change from 4C1 to 1,4B was observed. 相似文献
17.
Karen A. Lewis Danielle A. Pfaff Jennifer N. Earley Sarah E. Altschuler Deborah S. Wuttke 《Nucleic acids research》2014,42(1):475-484
Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions. 相似文献
18.
In computational structural biology, structure comparison is fundamental for our understanding of proteins. Structure comparison is, e.g., algorithmically the starting point for computational studies of structural evolution and it guides our efforts to predict protein structures from their amino acid sequences. Most methods for structural alignment of protein structures optimize the distances between aligned and superimposed residue pairs, i.e., the distances traveled by the aligned and superimposed residues during linear interpolation. Considering such a linear interpolation, these methods do not differentiate if there is room for the interpolation, if it causes steric clashes, or more severely, if it changes the topology of the compared protein backbone curves. To distinguish such cases, we analyze the linear interpolation between two aligned and superimposed backbones. We quantify the amount of steric clashes and find all self-intersections in a linear backbone interpolation. To determine if the self-intersections alter the protein’s backbone curve significantly or not, we present a path-finding algorithm that checks if there exists a self-avoiding path in a neighborhood of the linear interpolation. A new path is constructed by altering the linear interpolation using a novel interpretation of Reidemeister moves from knot theory working on three-dimensional curves rather than on knot diagrams. Either the algorithm finds a self-avoiding path or it returns a smallest set of essential self-intersections. Each of these indicates a significant difference between the folds of the aligned protein structures. As expected, we find at least one essential self-intersection separating most unknotted structures from a knotted structure, and we find even larger motions in proteins connected by obstruction free linear interpolations. We also find examples of homologous proteins that are differently threaded, and we find many distinct folds connected by longer but simple deformations. TM-align is one of the most restrictive alignment programs. With standard parameters, it only aligns residues superimposed within 5 Ångström distance. We find 42165 topological obstructions between aligned parts in 142068 TM-alignments. Thus, this restrictive alignment procedure still allows topological dissimilarity of the aligned parts. Based on the data we conclude that our program ProteinAlignmentObstruction provides significant additional information to alignment scores based solely on distances between aligned and superimposed residue pairs. 相似文献
19.
Reaction of Cerebratulus lacteus toxin B-IV with tetranitromethane in the presence of low concentrations of urea results in essentially complete loss of toxicity as measured by a sensitive quantal bioassay. Amino acid analysis and speetrophotometric studies both indicate the primary effect of reaction to be nitration of a single tyrosine residue per molecule of toxin. The nitrated residue has been identified as tyrosine-9 by automated Edman degradation of the modified protein. Since the secondary structure of toxin B-IV is not detectably altered by nitration, it is concluded that tyrosine-9 is directly involved in the interaction of this polypeptide with its axonal receptor, proposed to be involved in the inactivation of voltage-sensitive Na+ channels in crustacean nerves. 相似文献
20.
Ca2+ and other divalent cations can trigger aggregation of phospholipid vesicles containing phosphatidic acid or phosphatidylserine. The reaction, which can be detected by an increase in light scattering, has a critical dependence on the Ca2+ concentration, with a threshold near 4 mM Ca2+. This is the concentration for half-saturation of the polar head groups and for full neutralization of the membrane surface charge. The aggregation proceeds as a “polymerization” reaction, eventually forming such large aggregates that the vesicles precipitate. The stopped-flow rapid mixing technique was used to study the vesicle dimerization reaction which is the first step in the overall aggregation process. Vesicle dimerization resulted in a doubling of light scattering and had a vesicle concentration-dependent time constant () which varied between 0.4 and 2.0 s under the conditions of the study. Analysis of the dependence of the reaction amplitude and on the concentrations of vesicles and Ca2+ showed that the Ca2+ binding is fast, and that the dimerization proceeds by a mechanism in which the vesicles first collide to form an encounter complex followed by a slower conversion of the encounter complex to a stable complex. For phosphatidic acid vesicles, about 200–700 collisions are necessary to achieve a stable dimer. The rate-limiting step in the overall reaction in thus the transformation of the encounter complex into a stable complex, requiring 0.5 and 1.0 ms. The above-mentioned results are relatively insensitive to the type of divalent cation or to the choice of negatively charged lipid (phosphatidic acid or phosphatidylserine).Evidence is given that the stable complex is effected by Ca2+-mediated salt bridges between the two membranes and that the rate constant of the transformation step derives from the statistics of the distribution and the rate of redistribution of Ca2+-occupied polar head groups on the membrane surfaces. The relevance of these results to the problem of Ca2+-induced fusion of biological membranes is discussed. 相似文献