Oligomeric Structure Research Articles

DFT calculations of photophysical properties of ethylen-dioxy-thiophen derivatives oligomers with optoelectronic functionalities

Materials with π-conjugated system can absorb sunlight, create photo-generated current or even produce light. The easiness of its manipulation combined with cheap process has made organic components provide a new attractive field for electronic materials research. This paper presents computational study of π-conjugated oligomers structure. In this regard, we used density functional theory (DFT) calculations to analyze the geometry of oligomers based of EDOT (3,4-ethylen dioxythiophen) and VC (vinyl-carbazol). The band gap with simulated spectra (UV–Vis spectra, emission spectra) and several photovoltaic properties like open-circuit photovoltage (Voc) and light harvesting efficiency (LHE) are predicted and discussed for giving theoretical knowledge of studied compound efficacity in photovoltaic applications.

β-Carotene, a Potent Amyloid Aggregation Inhibitor, Promotes Disordered Aβ Fibrillar Structure

The aggregation of amyloid beta (Aβ) into fibrillar aggregates is a key feature of Alzheimer's disease (AD) pathology. β-carotene and related compounds have been shown to associate with amyloid aggregates and have direct impact on the formation of amyloid fibrils. However, the precise effect of β-carotene on the structure of amyloid aggregates is not known, which poses a limitation towards developing it as a potential AD therapeutic. In this report, we use nanoscale AFM-IR spectroscopy to probe the structure of Aβ oligomers and fibrils at the single aggregate level and demonstrate that the main effect of β-carotene towards modulating Aβ aggregation is not to inhibit fibril formation but to alter the secondary structure of the fibrils and promote fibrils that lack the characteristic ordered beta structure.

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties.

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

Molecular dynamics simulations reveal the importance of amyloid-beta oligomer β-sheet edge conformations in membrane permeabilization

Oligomeric aggregates of the amyloid-beta peptide(1-42) (Aβ42) are regarded as a primary cause of cytotoxicity related to membrane damage in Alzheimer's disease. However, a dynamical and structural characterization of pore-forming Aβ42 oligomers at atomic detail has not been feasible. Here, we used Aβ42 oligomer structures previously determined in a membrane-mimicking environment as putative model systems to study the pore formation process in phospholipid bilayers with all-atom molecular dynamics simulations. Multiple Aβ42 oligomer sizes, conformations, and N-terminally truncated isoforms were investigated on the multi-μs time scale. We found that pore formation and ion permeation occur via edge conductivity and exclusively for β-sandwich structures that feature exposed side-by-side β-strand pairs formed by residues 9-21 of Aβ42. The extent of pore formation and ion permeation depends on the insertion depth of hydrophilic residues 13-16 (HHQK domain) and thus on subtle differences in the overall stability, orientation, and conformation of the aggregates in the membrane. Additionally, we determined that backbone carbonyl and polar side-chain atoms from the edge strands directly contribute to the coordination sphere of the permeating ions. Furthermore, point mutations that alter the number of favorable side-chain contacts correlate with the ability of the Aβ42 oligomer models to facilitate ion permeation in the bilayer center. Our findings suggest that membrane-inserted, layered β-sheet edges are a key structural motif in pore-forming Aβ42 oligomers independent of their size and play a pivotal role in aggregate-induced membrane permeabilization.

Insights into the oligomeric structure of the HIV-1 Vpu protein

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in β-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.

A double point mutation of SOD1 targeting net charge promotes aggregation under destabilizing conditions: Correlation of charge distribution and ALS-provoking mutation

A progressive neurodegenerative illness such as amyotrophic lateral sclerosis (ALS) is characterized by the misfolding and aggregation of human CuZn superoxide dismutase (hSOD1) into amyloid aggregates. Thus, designing strategies for the choice of WT-SOD1 and double mutant (G12D/G138E) with an increased net negative charge can be a good idea to elucidate the pathological mechanism of SOD1 in ALS under some destabilizing conditions. Consequently, we show evidence that protein charge, together with other destabilizing conditions, plays an important role in ALS disease. To achieve this purpose, we use methods, such as spectroscopy and transmission electron microscopy (TEM) to monitor the formation of amyloid aggregation. The specific activity of WT-SOD1 was approximately 1.72 times higher than that of the double mutant. Under amyloidogenic circumstances, structural properties such as local, secondary, oligomeric, and fibrillar structures were explored. The double mutant's far-UV CD spectra displayed a broad minimum peak in the region 213 to 218 nm, suggesting the production of β-rich amyloid fibrils. FTIR spectra of the double mutant samples at different incubation times showed a low-frequency peak around 1630–1640 cm-1, attributed to a parallel β-sheet. Moreover, CR-binding assay and TEM analysis revealed and confirmed that mutation with an increased repulsive charge promotes the formation of fibrous aggregates. Consequently, ALS mutations with a higher repulsive charge are the apparent exceptions that validate the rule. This findings revealed that the double mutant increases protein aggregation through a novel mechanism, likely involving destabilization of structure and a change in the net negative charge.

Lipids uniquely alter the secondary structure and toxicity of amyloid beta 1-42 aggregates.

Abrupt aggregation of amyloid β1-42 (Aβ) peptide is a hallmark of Alzheimer's disease (AD), a severe pathology that affects more than 44million people worldwide. A growing body of evidence suggests that lipids can uniquely alter rates of Aβ1-42 aggregation. However, it remains unclear whether lipids only alter rates of protein aggregation or also uniquely modify the secondary structure and toxicity of Aβ1-42 oligomers and fibrils. In this study, we investigated the effect of phosphatidylcholine (PC), cardiolipin (CL), and cholesterol (Chol) on Aβ1-42 aggregation. We found that PC, CL and Chol strongly accelerated the rate of fibril formation compared to the rate of Aβ1-42 aggregation in the lipid-free environment. Furthermore, anionic CL enabled the strongest acceleration of Aβ1-42 aggregation compared to zwitterionic PC and uncharged Chol. We also found that PC, CL and Chol uniquely altered the secondary structure of early-, middle- and late-stage Aβ1-42 aggregates. Specifically, CL and Chol drastically increased the amount of parallel β-sheet in Aβ1-42 oligomers and fibrils grown in the presence of these lipids. This caused a significant increase in the toxicity of Aβ:CL and Aβ:Chol compared to the toxicity of Aβ:PC and Aβ1-42 aggregates formed in the lipid-free environment. These results demonstrate that toxicity of Aβ aggregates correlates with the amount of their β-sheet content, which, in turn, is determined by the chemical structure of lipids present at the stage of Aβ1-42 aggregation.

The cryo-EM structure of full-length RAD52 protein contains an undecameric ring.

The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double-strand break repair. The N-terminal half of RAD52 is primarily responsible for self-oligomerisation and DNA binding. Crystallographic studies have revealed the detailed structure of the N-terminal half. However, only low-resolution structures have been reported for the full-length protein, and thus the structural role of the C-terminal half in self-oligomerisation has remained elusive. In this study, we determined the solution structure of the human RAD52 protein by cryo-electron microscopy (cryo-EM), at an average resolution of 3.5 Å. The structure revealed an undecameric ring that is nearly identical to the crystal structures of the N-terminal half. The cryo-EM map for the C-terminal half was poorly defined, indicating that the region is intrinsically disordered. The present cryo-EM structure provides important insights into the mechanistic roles played by the N-terminal and C-terminal halves of RAD52 during DNA double-strand break repair.

Oligomeric state and chaperon activity of Hsp60.

Heat-shock proteins are capable to exert their stabilizing action against biomolecules aggregation through different mechanisms, which are generally related to their molecular weight. Similar to its bacterial homologous GroEl, Hsp60 is known to work as a folding machine with the assistance of co-chaperonin Hsp10 and ATP. However, recent experimental evidence suggested an alternative mechanism exploited by Hsp60 towards aggregation prone molecules. Indeed, we already observed that Hsp60 stabilized Aβ40 against fibrillization in the absence of ATP by recruiting early small aggregates of Ab, Here we investigate the mechanism exerted by Hsp60 against the amyloid fibrillization in relation with its oligomeric structure. To this purpose, the monomeric or tetradecameric form of the protein were isolated and their effect on beta-amyloid aggregation was tested separately. The structural stability of the two forms of Hsp60 was also investigated by differential scanning calorimetry (DSC), Light Scattering and Circular Dichroism. Results shown that the monomeric form is less stable but more effective against amyloid fibrillization. A major functionality related to a less ordered and stable structure is a common treat of proteins having intrinsically disordered regions (IDR). In Hsp60 contacts between subunits in the equatorial domain have been recognized as the region of moonlighting character. Their exposure to the solvent confers to monomeric Hsp60 the ability to interact with amyloid molecules at a major extent than that due to the plain increase of the total protein surface.

Insights into the structure-function relationship of the HIV-1 Vpu protein.

HIV-1 is the causative agent of HIV/AIDS. Annually, hundreds of thousands of people carrying the virus die from virus-related causes. Still, the means to cure the HIV-caused diseases are limited. Acquiring detailed knowledge about the viral mechanisms to modulate cellular processes is vastly needed for the development of efficient anti-viral treatments. We study the molecular mechanisms of HIV-1-host interactions, particularly the virus-encoded proteins with major roles in the virus lifecycle. The focus is on the Vpu protein, which forms ion-conducting homo-oligomers in cellular membranes but the protein monomer is also deemed physiologically relevant due its interaction with host proteins. However, the structure of full-length (FL) Vpu, required for structure-based drug design, has not been resolved. Recently, we made significant progress toward the understanding of FL Vpu quaternary structure. We used negative staining electron microscopy (nsEM) and size exclusion chromatography to characterize Vpu oligomerization. We generated a chimera protein of maltose binding protein fused to Vpu N-terminal (MBP-Vpu) to increase the protein size to aid EM experiments. We found that the MBP-Vpu forms a stable oligomer (likely pentamer) in solution, possibly driven by the hydrophobic effect and TM self-association. We further found that when transferred to the lipid environment, these oligomers restructured significantly: along with the pentamers, low-order oligomers were observed as well, or under specific lipid conditions in lyso lipid Vpu formed liner array-like aggregates. This emphasizes the role of lipid environment on Vpu oligomer organization. All these findings are novel and the observed Vpu quaternary structures are most likely physiologically relevant. Our ongoing cryo-EM experiments aim to solve the high-resolution structure of Vpu oligomer. The characterization of Vpu-drug binding is ongoing in our lab as well.

Comparing the structure and stability of diverse amyloid beta oligomers using fluorescence correlation spectroscopy.

Soluble oligomers of amyloid beta (Aβ) play a central role in Alzheimer's Disease pathology, however the detailed mechanisms of Aβ oligomer toxicity remain under investigation. One challenge in measuring oligomer size or cytotoxicity is the variability between published protocols that describe distinct oligomers, and the likely scenario that there are multiple pathological oligomers rather than a single disease-causing complex. Given the heavy use of dilution and incubation in these protocols, a systematic review covering how different oligomeric concentrations and preparation protocols affect oligomeric size and aggregation propensity is essential for standardizing assays of amyloid toxicity. To begin addressing this gap, we have systematically characterized and compared two distinct amyloid beta oligomers derived from either a homogeneous detergent or from cell media using fluorescence correlation spectroscopy (FCS). We observe temporal changes in size and stability of these oligomers over a wide range of concentrations and attempt to codify their similarities and differences. Significant changes between oligomer populations are observed over hours, with more diluted complexes falling apart. These observations also demonstrate that protein oligomer behavior is highly specific to their environments, which must be accounted for in biological assays, especially in the presence of cell media. Accounting for such discrepancies will also enable high-throughput screening of compounds that inhibit Aβ oligomer cytotoxicity with high reproducibility, which is a crucial component of drug discovery in the context of neurodegeneration and aging.

Towards cloud-enabled fully automated end-to-end single molecule imaging.

Single Molecule Super-resolution imaging has revolutionised understanding biological processes on the sub-100 nm scale. Our group developed Fluorophore Localisation Imaging with Photobleaching (FLImP), a super-resolution imaging method that can achieve <4 nm resolution. In concert with molecular dynamics simulations, FLImP was used to reveal the in-situ atomic resolution structures of ligand-bound EGFR oligomers at the plasma membrane of cells (Needham et al., Nat Comms 2016), and those of ligand-unbound dimers and oligomers on cells (Zanetti-Domingues et al., Nat Comms 2018), shedding new light on the mechanisms of EGFR auto-inhibition, phosphorylation and signalling.

Covalently bound humin-lignin hybrids as important novel substructures in organosolv spruce lignins

Organosolv lignins (OSLs) are important byproducts of the cellulose-centred biorefinery that need to be converted in high value-added products for economic viability. Yet, OSLs occasionally display characteristics that are unexpected looking at the lignin motifs present. Applying advanced NMR, GPC, and thermal analyses, isolated spruce lignins were analysed to correlate organosolv process severity to the structural details for delineating potential valorisations. Very mild conditions were found to not fractionate the biomass, causing a mix of sugars, lignin-carbohydrate complexes (LCCs), and corresponding dehydration/degradation products and including pseudo-lignins. Employing only slightly harsher conditions promote fractionation, but also formation of sugar degradation structures that covalently incorporate into the oligomeric and polymeric lignin structures, causing the isolated organosolv lignins to contain lignin-humin hybrid (HLH) structures not yet evidenced as such in organosolv lignins. These structures effortlessly explain observed unexpected solubility issues and unusual thermal responses, and their presence might have to be acknowledged in downstream lignin valorisation.

Multimeric assembly and dynamics of pore-forming lantibiotic peptide Nisin.

Multidrug resistance in bacteria has led to a dire need for the development of new antibiotics with novel modes of action which can evade existing bacterial defenses. By targeting Lipid-II, antibiotic peptides such as Nisin disrupt bacteria's ability to synthesize their cell wall. Lantibiotics bind to Lipid-II's pyrophosphate moiety rather than the more commonly targeted peptide moiety. This means that bacteria that develop resistance to commercial antibiotics should still be vulnerable to lantibiotics. In this study, we use atomic-scale molecular dynamics computational studies to model stable oligomeric pore structures of Nisin and investigate their dynamics and stability. We test a variety of conformations of Nisin pore structures and motifs to find a viable structure. We also explore the role that Lipid-II plays in the formation and in maintaining the stability of the pores as well as the role transmembrane potentials play in the nature of the pores, including the channel volume and the ability of Nisin pores to exhibit ion selectivity.

Quantification of lateral associations of achondroplasia in FGFR3.

Fibroblast growth factor receptor 3 (FGFR3) belongs to the receptor tyrosine kinase (RTK) family and is a key player in bone growth, tumor angiogenesis, and other developmental processes. Of particular interest is a substitution of glycine to arginine (G380R) in the transmembrane domain, which is implicated in human achondroplasia. The G380R mutation has been reported to stabilize FGFR3 dimers in the absence of ligand; however, recent work has suggested that the G380R mutation may be inducing the formation of FGFR3 oligomers that are larger than dimers. This has propelled us to re-evaluate the effect of the mutation on FGFR3 self-association in the plasma membrane of live cells using a quantitative fluorescence method, FSI-FRET, which reports on oligomer size, stability, and structure. This project aims to re-examine the self-association of both wild type and G380R mutant FGFR3 over physiologically relevant receptor concentrations and under different growth factor conditions using FSI-FRET.

Atomic insights into amyloid-induced membrane damage.

Amphipathic peptides can cause biological membranes to leak either by dissolving their lipid content via a detergent-like mechanism or by forming pores on the membrane surface. These modes of membrane damage have been related to the toxicity of amyloid peptides and to the activity of antimicrobial peptides. Here, we will discuss all-atom simulations in which membrane-bound amphipathic peptides self-assemble into beta-sheets that subsequently either form stable pores inside the bilayer or drag lipids out of the membrane surface. An analysis of these simulations shows that the acyl tail of lipids interact strongly with non-polar side chains of peptides deposited on the membrane. These strong interactions enable lipids to be dragged out of the bilayer by oligomeric structures accounting for detergent-like damage. They also disturb the orientation of lipid tails in the vicinity of peptides. These distortions are minimized around pore structures. We also show that membrane-bound beta-sheets become twisted with one of their extremities partially penetrating the lipid bilayer. This allows peptides on opposite leaflets to interact and form a long transmembrane beta-sheet, which initiates poration. In simulations, where peptides are deposited on a single leaflet, the twist in beta-sheets allows them to penetrate the membrane and form pores. In addition, our simulations show that fibril-like structures produce little damage to lipid membranes, as non-polar side chains in these structures are unavailable to interact with the acyl tail of lipids.

Peptide-based inhibitors of amyloid beta aggregation and toxicity.

Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease. Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Previous work has shown that seven overlapping fragments of Aβ1-42 can disrupt the process of fibril formation, possibly through intercalation into the growing aggregate. The potential neuroprotective properties of these fragments were not directly examined. In this project, Aβ1-42 was allowed to aggregate in aqueous buffer for 2, 4, or 24 h in the absence and presence of an Aβ fragment, at 1:2 Aβ1-42/fragment molar ratio. Neuronal PC12 cells were then incubated with the samples for 50 h before conducting a cell viability assay. Thioflavin-T fluorescence assays to detect the fibrillar form of Aβ1-42 were also conducted. There was an inverse correlation between the time span of Aβ1-42 aggregation and cellular toxicity, with the 2 h sample exhibiting the greatest level of toxicity. Aβ fragments displayed varying effects on the inhibition of Aβ1-42 fibrillogenesis and suppression of its toxicity. Three fragments completely neutralized the toxicity of the 2 h aggregate, while two others conferred weak cellular protection against Aβ1-42 toxicity. One fragment enhanced Aβ1-42 toxicity at all stages of aggregation. Every fragment disrupted fibril formation to some extent, but there was no obvious correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. This suggests that fragment intercalation into the growing fibril may block further aggregation but not the toxicity of oligomeric Aβ1-42. Fragments that block both Aβ1-42 aggregation and toxicity represent potential targets for therapeutic development.

High‐Performance Zinc‐Ion Battery Enabled by Tuning the Terminal Group and Chain Length of PEO‐based Oligomers

AbstractSide reactions and dendrite growth issues originated from free water seriously hamper the application of zinc‐ion batteries (ZIB). The electrolyte strategy of using a co‐solvent system has been proven to effectively improve the performance of ZIB. However, lack of thorough understanding of the co‐solvent structure on the performance of electrolytes hinders the rational design of co‐solvent electrolytes. Here, by studying how the terminal group and chain length of polyethylene oxide (PEO) based oligomers affect the electrochemical performance of ZIB, we obtained the optimized PEO oligomer structure and the underlying mechanism. As a result, the designed co‐solvent electrolyte enables the Zn|| NH4V4O10 full cell to gain a 98.5 % capacity retention over 9500 cycles at 10 A g−1. This work provides new insights into the inhibition of water‐related side reactions and further promote the development of electrolytes for ZIB and other metal batteries.