Oligomeric Structure Research Articles

Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells.

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

Structural insights into rapamycin-induced oligomerization of a FRB-FKBP fusion protein.

Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events.

Chemical and Solvent‐Based Recycling of High‐Performance Epoxy Thermoset Utilizing Imine‐Containing Secondary Amine Hardener

AbstractEpoxy thermosets are indispensable in various industrial applications due to their exceptional material properties. However, their limited recyclability poses a significant challenge for sustainability. To address this issue, integrating recyclable imine moieties into epoxy systems has emerged as a promising strategy. Despite recent advancements, challenges persist concerning the hydrolytic stability of imine‐based epoxy thermosets and the formation of irregular oligomeric structures upon depolymerization. In this study, these shortcomings are solved by designing a liquid imine monomer with secondary amine functionalities, which is subsequently reacted with commercially available epoxy monomers to produce high‐performance epoxy thermosets. Remarkably, the developed imine‐based epoxy system shows no sign of hydrolysis even under high‐temperature aqueous conditions, underscoring its potential for various industrial applications. Only the treatment with aqueous HCl results in the selective formation of exclusively well‐defined molecules, in high isolated yields after depolymerization. Crosslinking these building blocks once again yields recycled polymers with properties identical to pristine material. Moreover, it is discovered that, despite the crosslinked nature of the epoxy thermoset, the complete dissolution of the intact polymer in dichloromethane allowed for simple solvent‐based recycling. Through design on the molecular level, the recycling of imine‐based epoxy polymers is achieved via chemical and solvent‐based techniques.

De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure.

Recent attention has focused on the de novo design of proteins, paralleling advancements in biopharmaceuticals. Achieving protein designs with both structure and function poses a significant challenge, particularly considering the importance of quaternary structures, such as oligomers, in protein function. The cell penetration properties of peptides are of particular interest as they involve the penetration of large molecules into cells. We previously suggested a link between the oligomerization propensity of amphipathic peptides and their cell penetration abilities, yet concrete evidence at cellular-relevant concentrations was lacking due to oligomers' instability. In this study, we sought to characterize oligomerization states using various techniques, including X-ray crystallography, acceptor photobleaching Förster resonance energy transfer (FRET), native mass spectrometry (MS), and differential scanning calorimetry (DSC), while exploring the function related to oligomer status. X-ray crystallography revealed the atomic structures of oligomers formed by LK-3, a bis-disulfide bridged dimer with amino acid sequence LKKLCLKLKKLCKLAG, and its derivatives, highlighting the formation of hexamers, specifically the trimer of dimers, which exhibited a stable hydrophobic core. FRET experiments showed that LK-3 oligomer formation was associated with cell penetration. Native MS confirmed higher-order oligomers of LK-3, while an intriguing finding was the enhanced cell-penetrating capability of a 1:1 mixture of l/d-peptide dimers compared to pure enantiomers. DSC analysis supported the notion that this enantiomeric mixture promotes the formation of functional oligomers, crucial for cell penetration. In conclusion, our study provides direct evidence that amphipathic peptide LK-3 forms oligomers at low nanomolar concentrations, underscoring their significance in cell penetration behavior.

Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ∼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.

Interfering with aggregated α-synuclein in advanced melanoma leads to a major upregulation of MHC class II proteins.

Melanoma is the most serious and deadly form of skin cancer and with progression to advanced melanoma, the intrinsically disordered protein α-synuclein is upregulated to high levels. While toxic to dopaminergic neurons in Parkinson's disease, α-synuclein is highly beneficial for primary and metastatic melanoma cells. To gain detailed insights into this exact opposite role of α-synuclein in advanced melanoma, we performed proteomic studies of high-level α-synuclein-expressing human melanoma cell lines that were treated with the diphenyl-pyrazole small-molecule compound anle138b, which binds to and interferes with the oligomeric structure of α-synuclein. We also performed proteomic and transcriptomic studies of human melanoma xenografts that were treated systemically with the anle138b compound. The results reveal that interfering with oligomerized α-synuclein in the melanoma cells in these tumor xenografts led to a substantial upregulation and expression of major histocompatibility complex proteins, which are pertinent to enhancing anti-melanoma immune responses.

Self-assembled oligomeric structures of an asymmetric molecular linker; 4-isocyanophenyl disulfide on Au(111)

Para-substituted benzenes, such as 1,4-benzene dithiol and 1,4-phenyl diisocyanide, have been observed to oligomerize on the Au(111) surface by incorporating gold adatoms extracted from the substrate. This work investigates if oligomerization occurs for an analogous but asymmetric linker, 4-isocyanophenyl disulfide (ICPD) on Au(111). This molecule is comprised of both disulfide and isocyanide terminal groups attached to the phenyl ring. The resulting surface structures formed on Au(111) following exposure to ICPD are studied using scanning tunneling microscopy (STM). 1,4-isocyanophenyl thiolate (ICPT), formed through scission of ICPD’s disulfide bond, was also found to oligomerize on the surface, and potential oligomer structures and binding geometries are proposed with the aid of density functional theory (DFT) calculations, along with simulated STM images of the resulting structures. It is observed in this work that ICPT forms oligomeric structures that cover large sections of the substrate and appear to create etch pits resulting from gold atom extraction. Numerous potential binding geometries are investigated based on the distances between substrate gold atom adsorption sites compared to the monomer length. Selected structural candidates were optimized using DFT and were used to generate simulated STM images using the Tersoff–Hamann method to compare with experiment. It has been shown previously that the isocyanide- and thiol-connected oligomers conduct electrons, suggesting the possibility that the asymmetric oligomers found here might form the basis for fabricating molecular diodes.

Ir(I)-Bi(III) Donor-Acceptor Adducts Stabilized by Dispersion Interactions between the Metal Pincer Ligands and Their Possible Self-Assembly Forming Molecular 1D Semiconductors.

Structure, stability, and electronic properties of the bimetallic {[IrI(terpy)(Me)]-[BiIIINNN]}n monomeric, oligomeric, and polymeric structures (n = 1-3 and ∞; terpy = terpyridine; Me = methyl; BiNNN = bismuth triamide) and their derivatives (designated as (Bi·Ir)n structures) were studied theoretically by DFT cluster and periodic calculations. Stable Bi·Ir adducts (monomers) were formed with short Bi-Ir bonds (<2.7 Å) and Gibbs free binding energies larger than 20 kcal/mol for all systems. The substitution of the pincer ligands of Ir(I) and Bi(III) complexes by the electron-donating (NH2) and electron-withdrawing (NO2, F, CF3) groups, respectively, enhanced the Ir → Bi charge transfer, substantially stabilizing the Bi·Ir monomers. The monomers from the unsubstituted complexes can be considered as dispersion stabilized adducts, and they may form spontaneously (Bi·Ir)n layered oligomers/polymers with semiconducting properties. The self-assembly of monomers into oligomers/polymers is hindered by bulkier protecting groups on the Bi(III) complex, such as tBu and SiMe3.

Functional diversity in archaeal Hsp60: a molecular mosaic of Group I and Group II chaperonin.

External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium andensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP-dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, β, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αβγ, Hsp60αβ, and Hsp60β, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP-dependent manner through charge-driven interactions. Furthermore, the Hsp60β homo-oligomeric complex can protect the archaeal and eukaryotic membrane from stress-induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC-type cooperativity is nested within KNF-type cooperativity. Remarkably, during ATP hydrolysis, Hsp60β, and Hsp60αβ complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality.

A Theoretical Investigation into the Oligomer Structure of Carbon Dots Formed from Small-Molecule Precursors.

In-depth insights into the oligomers of carbon dots (CDs) prepared from small-molecule precursors are important in the study of the carbonization mechanism of CDs and for our knowledge of their complex structure. Herein, citric acid (CA) and ethylenediamine (EDA) were used as small-molecule precursors to prepare CDs in an aqueous solution. The structure of oligomers acquired from CA and EDA in different molar ratios and their formation process were first studied using density functional theory, including the dispersion correction (DFT-D3) method. The results showed that the energy barrier of dimer cyclization was higher than that of its linear polymerization, but the free energy of the cyclized product was much lower than that of its reactant, and IPCA (5-oxo-1,-2,3,5-tetrahydroimidazo [1,2-a]pyridine-7-carboxylic acid) could therefore be obtained under certain conditions. The oligomers obtained from different molar ratios of EDA and CA were molecular clusters formed by short polyamide chains through intermolecular forces; with the exception of when the molar ratio of EDA to CA was 0.5, excessive CA did not undergo an amidation reaction but rather attained molecular clusters directly through intermolecular forces. These oligomers exhibited significant differences in their surface functional groups, which would affect the carbonization process and the surface structure of CDs.

Aggregation Mechanisms and Molecular Structures of Amyloid-β in Alzheimer's Disease.

Amyloid plaques are a major pathological hallmark involved in Alzheimer's disease and consist of deposits of the amyloid-β peptide (Aβ). The aggregation process of Aβ is highly complex, which leads to polymorphous aggregates with different structures. In addition to aberrant aggregation, Aβ oligomers can undergo liquid-liquid phase separation (LLPS) and form dynamic condensates. It has been hypothesized that these amyloid liquid droplets affect and modulate amyloid fibril formation. In this review, we briefly introduce the relationship between stress granules and amyloid protein aggregation that is associated with neurodegenerative diseases. Then we highlight the regulatory role of LLPS in Aβ aggregation and discuss the potential relationship between Aβ phase transition and aggregation. Furthermore, we summarize the current structures of Aβ oligomers and amyloid fibrils, which have been determined using nuclear magnetic resonance (NMR) and cryo-electron microscopy (cryo-EM). The structural variations of Aβ aggregates provide an explanation for the different levels of toxicity, shed light on the aggregation mechanism and may pave the way towards structure-based drug design for both clinical diagnosis and treatment.

PUResNetV2.0: a deep learning model leveraging sparse representation for improved ligand binding site prediction

Accurate ligand binding site prediction (LBSP) within proteins is essential for drug discovery. We developed ProteinUNetResNetV2.0 (PUResNetV2.0), leveraging sparse representation of protein structures to improve LBSP accuracy. Our training dataset included protein complexes from 4729 protein families. Evaluations on benchmark datasets showed that PUResNetV2.0 achieved an 85.4% Distance Center Atom (DCA) success rate and a 74.7% F1 Score on the Holo801 dataset, outperforming existing methods. However, its performance in specific cases, such as RNA, DNA, peptide-like ligand, and ion binding site prediction, was limited due to constraints in our training data. Our findings underscore the potential of sparse representation in LBSP, especially for oligomeric structures, suggesting PUResNetV2.0 as a promising tool for computational drug discovery.

Nucleic acid mediated activation of a short prokaryotic Argonaute immune system

A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P)+ hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15–3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection.

Characterization of Excited-State Electronic Structure in Diblock π-Conjugated Oligomers with Adjustable Linker Electronic Coupling.

Diblock conjugated oligomers are π-conjugated molecules that contain two segments having distinct frontier orbital energies and HOMO-LUMO gap offsets. These oligomers are of fundamental interest to understand how the distinct π-conjugated segments interact and modify their excited state properties. The current paper reports a study of two series of diblock oligomers that contain oligothiophene (Tn) and 4,7-bis(2-thienyl)-2,1,3-benzothiadiazole (TBT) segments that are coupled by either ethynyl (-C≡C-) or trans-(-C≡C-)2Pt(II)(PBu3)2 acetylide linkers. In these structures, the Tn segment is electron rich (donor), and the TBT is electron poor (acceptor). The diblock oligomers are characterized by steady-state and time-resolved spectroscopy, including UV-visible absorption, fluorescence, fluorescence lifetimes, and ultrafast transient absorption spectroscopy. Studies are compared in several solvents of different polarity and with different excitation wavelengths. The results reveal that the (-C≡C-) linked oligomers feature a delocalized excited state that takes on a charge transfer (CT) character in more polar media. In the (-C≡C-)2Pt(II)(PBu3)2-linked oligomers, there is weak coupling between the Tn and TBT segments. Consequently, short wavelength excitation selectively excites the Tn segment, which then undergoes ultrafast energy transfer (~1 ps) to afford a TBT-localized excited state.

Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2

The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs associate into oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the stability of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted, and point to a more complex role of the TM helix than the one expected from the “TM dimer switch” model.

Revealing the Effect of Stereocontrol on Intermolecular Interactions between Abiotic, Sequence-Defined Polyurethanes and a Ligand.

The development of precision polymer synthesis has facilitated access to a diverse library of abiotic structures wherein chiral monomers are positioned at specific locations within macromolecular chains. These structures are anticipated to exhibit folding characteristics similar to those of biotic macromolecules and possess comparable functionalities. However, the extensive sequence space and numerous variables make selecting a sequence with the desired function challenging. Therefore, revealing sequence-function dependencies and developing practical tools are necessary to analyze their conformations and molecular interactions. In this study, we investigate the effect of stereochemistry, which dictates the spatial location of backbone and pendant groups, on the interaction between sequence-defined oligourethanes and bisphenol A ligands. Various methods are explored to analyze the receptor-like properties of model oligomers and the ligand. The accuracy of molecular dynamics simulations and experimental techniques is assessed to uncover the impact of discrete changes in stereochemical arrangements on the structures of the resulting complexes and their binding strengths. Detailed computational investigations providing atomistic details show that the formed complexes demonstrate significant structural diversity depending on the sequence of stereocenters, thus affecting the oligomer-ligand binding strength. Among the tested techniques, the fluorescence spectroscopy data, fitted to the Stern-Volmer equation, are consistently aligned with the calculations, thus validating the developed simulation methodology. The developed methodology opens a way to engineer the structure of sequence-defined oligomers with receptor-like functionality to explore their practical applications, e.g., as sensory materials.

Primary Nucleation of Polymorphic α-Synuclein Dimers Depends on Copper Concentrations and Definite Copper-Binding Site.

The primary nucleation process of α-synuclein (AS) that forms toxic oligomeric species is the early stage of the pathological cause of Parkinson's disease. It is well-known that copper influences this primary nucleation process. While significant efforts have been made to solve the structures of polymorphic AS fibrils, the structures of AS oligomers and the copper-bound AS oligomers at the molecular level and the effect of copper concentrations on the primary nucleation are elusive. Here, we propose and demonstrate new molecular mechanism pathways of primary nucleation of AS that are tuned by distinct copper concentrations and by a specific copper-binding site. We present the polymorphic AS dimers bound to different copper-binding sites at the atomic resolution in high- and low-copper concentrations, using extensive molecular dynamics simulations. Our results show the complexity of the primary nucleation pathways that rely on the copper concentrations and the copper binding site. From a broader perspective, our study proposes a new strategy to control the primary nucleation of other toxic amyloid oligomers in other neurodegenerative diseases.

Heat-activated growth of metastable and length-defined DNA fibers expands traditional polymer assembly

Biopolymers such as nucleic acids and proteins exhibit dynamic backbone folding, wherein site-specific intramolecular interactions determine overall structure. Proteins then hierarchically assemble into supramolecular polymers such as microtubules, that are robust yet dynamic, constantly growing or shortening to adjust to cellular needs. The combination of dynamic, energy-driven folding and growth with structural stiffness and length control is difficult to achieve in synthetic polymer self-assembly. Here we show that highly charged, monodisperse DNA-oligomers assemble via seeded growth into length-controlled supramolecular fibers during heating; when the temperature is lowered, these metastable fibers slowly disassemble. Furthermore, the specific molecular structures of oligomers that promote fiber formation contradict the typical theory of block copolymer self-assembly. Efficient curling and packing of the oligomers – or ‘curlamers’ – determine morphology, rather than hydrophobic to hydrophilic ratio. Addition of a small molecule stabilises the DNA fibers, enabling temporal control of polymer lifetime and underscoring their potential use in nucleic-acid delivery, stimuli-responsive biomaterials, and soft robotics.

Enzymatic Synthesis and Structural Modeling of Bio-Based Oligoesters as an Approach for the Fast Screening of Marine Biodegradation and Ecotoxicity.

Given the widespread use of esters and polyesters in products like cosmetics, fishing nets, lubricants and adhesives, whose specific application(s) may cause their dispersion in open environments, there is a critical need for stringent eco-design criteria based on biodegradability and ecotoxicity evidence. Our approach integrates experimental and computational methods based on short oligomers, offering a screening tool for the rapid identification of sustainable monomers and oligomers, with a special focus on bio-based alternates. We provide insights into the relationships between the chemical structure and properties of bio-based oligomers in terms of biodegradability in marine environments and toxicity in benchmark organisms. The experimental results reveal that the considered aromatic monomers (terephthalic acid and 2,5-furandicarboxylic acid) accumulate under the tested conditions (OECD 306), although some slight biodegradation is observable when the inoculum derives from sites affected by industrial and urban pollution, which suggests that ecosystems adapt to non-natural chemical pollutants. While clean seas are more susceptible to toxic chemical buildup, biotic catalytic activities offer promise for plastic pollution mitigation. Without prejudice to the fact that biodegradability inherently signifies a desirable trait in plastic products, nor that it automatically grants them a sustainable "license", this study is intended to facilitate the rational design of new polymers and materials on the basis of specific uses and applications.

Cytotoxic Effect of Amyloid-β1-42 Oligomers on Endoplasmic Reticulum and Golgi Apparatus Arrangement in SH-SY5Y Neuroblastoma Cells.

Amyloid-β oligomers are a cytotoxic structure that is key for the establishment of the beginning stages of Alzheimer's disease (AD). These structures promote subcellular alterations that cause synaptic dysfunction, loss of cell communication, and even cell death, generating cognitive deficits. The aim of this study was to investigate the cytotoxic effects of amyloid-β1-42 oligomers (AβOs) on the membranous organelles involved in protein processing: the endoplasmic reticulum (ER) and Golgi apparatus (GA). The results obtained with 10 μM AβOs in SH-SY5Y neuroblastoma cells showed that oligomeric structures are more toxic than monomers because they cause cell viability to decrease as exposure time increases. Survivor cells were analyzed to further understand the toxic effects of AβOs on intracellular organelles. Survivor cells showed morphological alterations associated with abnormal cytoskeleton modification 72-96 h after exposure to AβOs. Moreover, the ER and GA presented rearrangement throughout the cytoplasmic space, which could be attributed to a lack of constitutive protein processing or to previous abnormal cytoskeleton modification. Interestingly, the disorganization of both ER and GA organelles exposed to AβOs is likely an early pathological alteration that could be related to aberrant protein processing and accumulation in AD.