Single Trimer Research Articles

Production and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: Insights from multi-angle light scattering.

The global threat of pandemics highlights the urgency of developing innovative vaccine strategies. Viral spike proteins are the primary antigens recognized by the immune system and serve as key targets for vaccine development. This study reports the production of full-length Influenza A virus surface glycoprotein, hemagglutinin (HA), and its incorporation into Nanodiscs (NDs). HA was expressed in insect cells and purified using detergents, maintaining its functional integrity. Characterization by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirmed that HA could be incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) NDs as a single trimer. SEC-MALS was instrumental in analysing the composition of NDs, which included HA, membrane scaffold proteins, lipids, and glycans. These findings provide a robust framework for the production and reconstitution of glycoproteins in NDs, and offers valuable insights into the study of multi-component nanoparticles using MALS. Our work highlights the potential of NDs for studying viral glycoproteins and advances the development of well-defined recombinant ND-based vaccines.

Ir noble metal atoms/clusters on graphene support: Insights from aberration corrected-STEM and density functional theory

The interactions of Iridium (Ir) noble metal atoms/clusters (Ir NMAs/NMCs) with graphene are investigated due to their remarkable catalytic performance, resulting from the use of 100 % metal atoms compared to nanoparticle counterparts. Using scanning transmission electron microscopy (STEM), various forms of Ir dispersed on graphene were observed, including single atoms, dimers, and trimers clusters. The STEM data further revealed inter-atomic distances of 2.83 Å and 2.96 Å for dimers and trimers which were 20 % and 17 % larger than the numbers found through density functional theory (DFT) calculations. The DFT calculations further showed that the Dirac-cone of graphene at Ir NMAs/NMCs locations changes to metallic form due to the emergence of energy states at the Fermi level. This effect results in non-zero effective masses of charge carriers but enhances their density of states (DOS) by 2-fold which is believed to increase the energy absorption efficiency of graphene in the infra-red range of radiation.

Charge State of Au Atoms on an Oxidized Rutile TiO2(110) Surface by AFM/KPFM at 78 K.

The charge state of noble metal atoms on a semiconductor surface is an important factor in surface catalysis. In this study, Au atoms were deposited on the rutile TiO2(110) surface to characterize its charge properties using atomic force microscopy with Kelvin probe force microscopy at 78 K. Au single atoms, dimers, and trimers at different sites on the surface were investigated. Positively charged Au atoms were verified at oxygen sites, while negatively charged Au atoms were found near oxygen vacancy sites. Furthermore, the charge states of small Au nanoclusters were clarified. Understanding the charge states of Au atoms is significant for identifying their efficient catalytic effects in surface catalysis.

Annexin proteins involved in plasma membrane repair reshape a membrane edge: Direct evidence from molecular simulations.

The Annexin (ANX) family of Ca+2-dependent membrane-binding proteins are implicated in plasma membrane repair in animal cells. The inward flux of Ca+2 upon formation of a plasma membrane lesion/pore drives the binding of cytoplasmic ANX proteins to the membrane, close to the pore. Annexins are deemed responsible for plasma membrane repair and are overexpressed in cancer cells where the plasma membrane is in the need for frequent repair. Annexin A5 (ANXA5) trimers form crystalline arrays solid-supported membranes (1), and owing to their concave disc-like shape, induce negative curvature on a flat plasma membrane (2), and are therefore likely to reshape the membrane in the vicinity of the membrane pore to support membrane repair (3). However, no direct evidence of membrane shaping has yet emerged. Here, we simulate ANXA5 binding on a bicelle, which models a membrane pore under periodic boundary conditions, and demonstrate that increasing concentration of ANXA5 trimers induces a monotonically increasing out-of-plane upward curvature in the bicelle. Interestingly, this overall curvature amounts to half the curvature induced by a single ANXA5 trimer on a flat lipid bilayer. The out of plane curvature induced by ANXA5 trimers near a membrane pore is demonstrated for the first time. Our results have important implications in the membrane-reshaping properties of Annexins and their ability to repair membranes.

Recently Designed Multivalent Spike Binders Cannot Bind Multivalently─How Do They Achieve Enhanced Avidity to SARS-CoV-2?

The trimeric spike protein of SARS-CoV-2 has been targeted by antibody mimics that bind near or at the receptor-binding domain to neutralize the virus. Several independent studies have reported enhanced binding avidity for dimers and trimers, where binding domains are connected by short peptides. The enhanced avidity of the multivalent constructs was attributed to their simultaneously binding two or three sites within a single spike trimer. We argue here that the 15-20 amino acid peptide linkers, when considered as worm-like-chains, are too short to span the binding sites within a single spike. The enhanced avidity of the multivalent constructs may be explained by a rebinding mechanism, which does not involve multivalent binding.

Near field FEM simulations of plasmonic gold nanoparticle based SERS substrate with experimental validation

Surface enhanced Raman spectroscopy or surface enhanced Raman scattering (SERS) being a very powerful technique for quantitative and qualitative analysis finds application in low concentration detection of chemical and biological samples. In this work, we have presented a complete methodology of simulating nanoparticles-based SERS substrate. Different configurations namely single nanoparticle, dimer, trimer and tetramer nanoparticle clusters are simulated and enhancement factor for each configuration is estimated. Presented simulations are carried out with COMSOL Multiphysics software using Electromagnetic Waves module (Wave Optics) in frequency domain. The model of gold nanoparticle is established to determine the scatter field, which was further used to estimate the enhancement factor. Simulation results thus obtained are then validated with experimental results. The simulation study using the COMSOL Multiphysics tool helps in the design and development of the SERS substrate for maximum electromagnetic enhancement at a particular wavelength. The presented simulation methodology will be helpful for various SERS related computational studies in future. Presented approach helps in understanding the effect of various parameters such as particle size, inter particle gap, excitation wavelength, etc. on the EM enhancement. Simulating various possibilities before fabrication helps in saving money and time. For device realization, gold nanoparticles are synthesized using Turkevich method to achieve uniform and spherical nanoparticles. Synthesized nanoparticles (40–45 nm radius) were drop casted on a porous cellulose paper substrate to realize a SERS substrate. Nanogaps created between nanoparticles become the hot-spot locations leading to electromagnetic coupling and hence the Raman signal enhancement. Paper based SERS substrate is tested with Rhodamine B (1 μM) test sample. Enhancement of 106 was observed with the developed SERS substrate. UV–Vis spectrometer and Raman spectrometers are used for various characterizations.

Synthesis of Novel Glycolipid Mimetics of Heparan Sulfate and Their Application in Colorectal Cancer Treatment in a Mouse Model.

Heparan sulfate (HS) is a highly sulfated natural carbohydrate that plays crucial roles in cancer, inflammation, and angiogenesis. Heparanase (HPSE) is the sole HS degrading endoglycosidase that cleaves HS at structure‐dependent sites along the polysaccharide chain. Overexpression of HPSE by cancer cells correlates with increased tumor size and enhanced metastasis. Previously we have shown that a tetramer HS mimetic is a potent HPSE inhibitor displaying remarkable anticancer activity in vivo. Building on that work, we report the synthesis and testing of a novel library of single entity trimer glycolipid mimetics that effectively inhibit HPSE at low nanomolar concentrations. A lipophilic arm was introduced to assess whether an improvement of pharmacokinetics and plasma residence time would offset the reduction in charge and multivalency. Preclinical tests in a mouse syngeneic model showed effective tumor growth inhibition by the tetramer but not the trimer glycomimetic.

A density functional theory study on the catalytic performance of metal (Ni, Pd) single atom, dimer and trimer for H2 dissociation

• H 2 dissociation on transition metal (TM = Ni, Pd) single atom, dimer and trimer doped on Au substrate have been studied. • The H 2 dissociation in the form of Ni, Pd trimer exhibited the best catalytic performance. • The H 2 dissociation process follow the BEP relation on different structural units. Elucidating the effect of different structural units in the catalyst on catalytic performance was very important for the development of the excellent heterogeneous catalyst. This work used density functional theory (DFT) approach to investigate H 2 dissociation over transition metal (TM = Ni, Pd) single atom, dimer and trimer doped on Au substrate. The influence of different structural units toward the catalytic performance was elucidated. The results showed that the catalytic performance of Ni, Pd trimer was stronger than those of Ni, Pd single atom and dimer. Therefore, it was proposed that for the H 2 dissociation, the structural units in the form of Ni, Pd trimer exhibited the best catalytic performance. It was also obtained that the H 2 dissociation process followed the BEP relation on different structural units. This work provides insights about structural units for the reasonable design of the excellent heterogeneous catalysts toward dissociation reactions.

The effect of (H2O)n (n = 1-3) clusters on the reaction of HONO with HCl: a mechanistic and kinetic study.

The reaction between HONO and HCl is a possible pathway for the generation of ClNO, which is prone to photolyze, produce chlorine radicals, and accelerate the oxidation of tropospheric VOCs. Current experimental and theoretical studies have significant differences in rate constants under similar conditions. This study aims to examine the reasons for this difference. In this study, the effects of a single water molecule, water dimer, water trimer, excess HCl and excess HONO on the reaction mechanism of HONO + HCl were studied at the CCSD(T)/aug-cc-pVTZ//M06-2X/6-311+G(2df,2p) level and the rate constants of each reaction channel were calculated. Our results showed that the reaction potential barrier of HONO with HCl was the lowest only when the water dimer was present, and the reaction rate constants were close to the experimental results, and both the cis-HONO⋯(H2O)2 + HCl and the trans-HONO⋯(H2O)2 + HCl reaction paths are likely to occur. We think that the reason for the inconsistency between experimental and theoretical results is that the water dimer is involved in the reaction in experiments.

Polarization-independent anapole response of a trimer-based dielectric metasurface

Abstract The phenomenon of anapole has attracted considerable attention in the field of metamaterials as a possible realization of radiationless objects. We comprehensively study this phenomenon in the cluster-based systems of dielectric particles by considering conditions of anapole manifestation in both single trimers of disk-shaped particles and metamaterial composed on such trimers. Our analytical approach is based on the multipole decomposition method and the secondary multipole decomposition technique. They allow us to associate the anapole with the multipole moments of the trimer and the separate multipole moments of its constitutive particles. The manifestation of anapole in a two-dimensional metamaterial (metasurface) is confirmed by checking the resonant states in the reflected field as well as from the electromagnetic near-field patterns obtained from the full-wave numerical simulation. It is demonstrated that the anapole excitation in trimers results in the polarization-independent suppression of reflection with the resonant enhancement of local electromagnetic fields in the metasurface. Finally, experimental verification of the theoretical results is presented and discussed.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations

Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.

A novel single-domain antibody multimer that potently neutralizes tetanus neurotoxin

Tetanus antitoxin, produced in animals, has been used for the prevention and treatment of tetanus for more than 100 years. The availability of antitoxins, ethical issues around production, and risks involved in the use of animal derived serum products are a concern. We therefore developed a llama derived single-domain antibody (VHH) multimer to potentially replace the conventional veterinary product. In total, 28 different tetanus neurotoxin (TeNT) binding VHHs were isolated, 14 of which were expressed in yeast for further characterization. Four VHH monomers (T2, T6, T15 and T16) binding TeNT with high affinity (KD < 1 nM), covering different antigenic domains as revealed by epitope binning, and including 3 monomers (T6, T15 and T16) that inhibited TeNT binding to neuron gangliosides, were chosen as building blocks to generate 11 VHH multimers. These multimers contained either 1 or 2 different TeNT binding VHHs fused to 1 VHH binding to either albumin (A12) or immunoglobulin (G13) to extend serum half-life in animals. Multimers consisting of 2 TeNT binding VHHs showed more than a 10-fold increase in affinity (KD of 4–23 pM) when compared to multimers containing only one TeNT binding VHH. The T6 and T16 VHHs showed synergistic in vivo TeNT neutralization and, when incorporated into a single VHH trimer (T6T16A12), they showed a very high TeNT neutralizing capacity (1,510 IU/mg).

Protein Extraction Efficiency and Selectivity of Esterified Styrene-Maleic Acid Copolymers in Thylakoid Membranes.

Amphiphilic styrene-maleic acid copolymers (SMAs) have been shown to effectively extract membrane proteins surrounded by an annulus of native membrane lipids via the formation of nanodiscs. Recent reports have shown that 2-butoxyethanol-functionalized SMA derivatives promote the extraction of membrane proteins from thylakoid membranes, whereas unfunctionalized SMA is essentially ineffective. However, it is unknown how the extent of functionalization and identity of sidechains impact protein solubilization and specificity. Herein, we show that the monoesterification of an SMA polymer with hydrophobic alkoxy ethoxylate sidechains leads to an increased solubilization efficiency (SE) of trimeric photosystem I (PSI) from the membranes of cyanobacterium Thermosynechococcus elongatus. The specific SMA polymer used in this study, PRO 10235, cannot encapsulate single PSI trimers from this cyanobacterium; however, as it is functionalized with alkoxy ethoxylates of increasing alkoxy chain length, a clear increase in the trimeric PSI SE is observed. Furthermore, an exponential increase in the SE is observed when >50% of the maleic acid repeat units are monoesterified with long alkoxy ethoxylates, suggesting that the PSI extraction mechanism is highly dependent on both the number and length of the attached side chains.

Symmetry analysis of trimer-based all-dielectric metasurfaces with toroidal dipole modes

Herein, we discuss the conditions for excitation of symmetry-protected toroidal dipole modes in all-dielectric metasurfaces composed of trimer or twin-trimer clusters of dielectric disks. Such metasurfaces permit enhanced light–matter interaction due to spatially confined light in resonant systems with a high-quality factor. To describe characteristics of toroidal modes existing in the clusters, we use the magnetic dipole moments approximation, group-theoretical methods, group representation theory, symmetry-adapted linear combination method, and circuit theory. To validate the obtained theoretical results, we fulfill both full-wave numerical simulations and microwave experiments. In particular, we have shown that the toroidal dipole mode appears as a quasi-dark state of the trimer. It can be excited in the metasurface by the field of a linearly polarized wave, providing the symmetry of the trimer is properly reduced. In the metasurface, the properties of the toroidal dipole mode are determined primarily by the parameters of a single trimer and are not a consequence of the periodicity of the array. The coupling of the toroidal dipole modes in the twin-trimers can appear in both bonding and anti-bonding fashion resulting in different orders of the net toroidal dipole moment. Due to the unique field configuration of these modes, the proposed metasurfaces can be considered as a platform for efficient light–matter interaction for enhanced absorption, non-linear switching, and sensing.

Chemo- and Stereospecific Solid-State Thermal Dimerization of Sodium trans-2-Butenoate and γ-Ray-Induced Single-Crystal-to-Single-Crystal Dimerization of Hexaaquamagnesium trans-2-Butenoate Dihydrate: Both Give rel-(3S,4R)-1-Hexene-3,4-dicarboxylate but by Different Mechanisms. Stereospecific γ-Ray-Induced Trimerization of Sodium trans-2-Butenoate

γ-Irradiation of crystalline hexaaquamagnesium trans-2-butenoate dihydrate affords rel-(3S,4R)-1-hexene-3,4-dicarboxylate by a single-crystal-to-single-crystal reaction. The reaction proceeds by a radical chain mechanism with anti addition to the butenoate double bond, as established by deuterium labeling. The product structure is that expected from the orientation of trans-2-butenoates in the pristine crystal. The same dicarboxylate is formed by heating crystalline sodium trans-2-butenoate at 300 °C, but the thermal ene reaction was shown by deuterium labeling to proceed by syn addition to the butenoate double bond as expected for a concerted ene reaction. γ-Irradiation of sodium trans-2-butenoate forms a single trimer chemo-, regio-, and stereospecifically. The structure of sodium trans-2-butenoate was determined, and the crystal packing is consistent with both the observed ene dimerization and γ-ray-induced trimerization.

Neutralizing Antibody Responses Induced by HIV-1 Envelope Glycoprotein SOSIP Trimers Derived from Elite Neutralizers.

The induction of broadly neutralizing antibodies (bNAbs) is a major goal in vaccine research. HIV-1-infected individuals that develop exceptionally strong bNAb responses, termed elite neutralizers, can inform vaccine design by providing blueprints for the induction of similar bNAb responses. We describe a new recombinant native-like envelope glycoprotein (Env) SOSIP trimer, termed AMC009, based on the viral founder sequences of an elite neutralizer. The subtype B AMC009 SOSIP protein formed stable native-like trimers that displayed multiple bNAb epitopes. Overall, its structure at 4.3-Å resolution was similar to that of BG505 SOSIP.664. The AMC009 trimer resembled one from a second elite neutralizer, AMC011, in having a dense and complete glycan shield. When tested as immunogens in rabbits, the AMC009 trimers did not induce autologous neutralizing antibody (NAb) responses efficiently while the AMC011 trimers did so very weakly, outcomes that may reflect the completeness of their glycan shields. The AMC011 trimer induced antibodies that occasionally cross-neutralized heterologous tier 2 viruses, sometimes at high titer. Cross-neutralizing antibodies were more frequently elicited by a trivalent combination of AMC008, AMC009, and AMC011 trimers, all derived from subtype B viruses. Each of these three individual trimers could deplete the NAb activity from the rabbit sera. Mapping the polyclonal sera by electron microscopy revealed that antibodies of multiple specificities could bind to sites on both autologous and heterologous trimers. These results advance our understanding of how to use Env trimers in multivalent vaccination regimens and the immunogenicity of trimers derived from elite neutralizers.IMPORTANCE Elite neutralizers, i.e., individuals who developed unusually broad and potent neutralizing antibody responses, might serve as blueprints for HIV-1 vaccine design. Here, we studied the immunogenicity of native-like recombinant envelope glycoprotein (Env) trimers based on viral sequences from elite neutralizers. While immunization with single trimers from elite neutralization did not recapitulate the breadth and potency of neutralization observed in these infected individuals, a combination of three subtype B Env trimers from elite neutralizers resulted in some neutralization breadth within subtype B viruses. These results should guide future efforts to design vaccines to induce broadly neutralizing antibodies.

A Protein Environment-Modulated Energy Dissipation Channel in LHCII Antenna Complex.

SummaryThe major light-harvesting complex of photosystem II (LHCII) is the main contributor to sunlight energy harvesting in plants. The flexible design of LHCII underlies a photoprotective mechanism whereby this complex switches to a dissipative state in response to high light stress, allowing the rapid dissipation of excess excitation energy (non-photochemical quenching, NPQ). In this work, we locked single LHCII trimers in a quenched conformation after immobilization of the complexes in polyacrylamide gels to impede protein interactions. A comparison of their pigment excited-state dynamics with quenched LHCII aggregates in buffer revealed the presence of a new spectral band at 515 nm arising after chlorophyll excitation. This is suggested to be the signature of a carotenoid excited state, linked to the quenching of chlorophyll singlet excited states. Our data highlight the marked sensitivity of pigment excited-state dynamics in LHCII to structural changes induced by the environment.

Enhancing Spectral Reflection through Controlled Phase Distribution Using Doped Polar-Dielectric Metasurfaces.

Controlling the phase distribution of wavefronts using optical metasurfaces has led to interesting optical properties and applications. Here, we explore the control of phase distribution through polar-dielectric metasurfaces composed of doped SiC nanosphere arrays. We investigate the impact of doping concentration on the optical properties of SiC nano-spheres. Our results indicate that increasing the doping of SiC nanoparticles influenced electric dipolar resonances, whereas it did not change the dipolar resonances. Using this concept, we numerically studied the extension of this idea to form metasurface arrays of single, dimer and linear trimers of the doped SiC nano-spheres. Using different doping schemes, we studied the improvement of the reflectivity at frequencies greater than the longitudinal optical phonon frequency.

A Bispecific Antibody That Simultaneously Recognizes the V2- and V3-Glycan Epitopes of the HIV-1 Envelope Glycoprotein Is Broader and More Potent than Its Parental Antibodies.

Broadly neutralizing antibodies (bNAbs) can prevent and control an HIV-1 infection, but their breadth is invariably too limited for use as monotherapy. To address this problem, bi- and trispecific antibody-like constructs have been developed. These engineered antibodies typically have greater breadth than the native bNAbs from which they were derived, but they are not more potent because they do not, in most cases, simultaneously engage more than a single epitope of the HIV-1 envelope glycoprotein (Env). Here, we describe a new class of bispecific antibodies targeting the V2-glycan (apex) and V3-glycan regions of the HIV-1 envelope glycoprotein (Env). Specifically, bispecific antibodies with a single-chain (scFv) form of the CAP256.VRC26.25 V2-glycan (apex) antibody on one antibody arm and a full V3-glycan Fab on the other arm neutralizes more HIV-1 isolates than the bNAbs from which they were derived. Moreover, these bispecific antibodies are markedly more potent than their parental bNAbs, likely because they simultaneously engage both the apex and V3-glycan epitopes of Env. Our data show that simultaneous engagement of two critical epitopes of a single Env trimer can markedly increase the potency of a bispecific antibody.IMPORTANCE Broadly neutralizing antibodies (bNAbs) can prevent a new HIV-1 infection and can at least temporarily suppress an established infection. However, antibody-resistant viruses rapidly emerge in infected persons treated with any single bNAb. Several bispecific antibodies have been developed to increase the breadth of these antibodies, but typically only one arm of these bispecific constructs binds the HIV-1 envelope glycoprotein trimer (Env). Here, we develop and characterize bispecific constructs based on well-characterized V2-glycan and V3-glycan bNAbs and show that at least one member of this class is more potent than its parental antibodies, indicating that they can simultaneously bind both of these epitopes of a single Env trimer. These data show that bispecific antibody-like proteins can achieve greater neutralization potency than the bNAbs from which they were derived.

A Flexible LHCII Structure Allows for Fine-Tuning of Excitation Energy Dissipation

The major light-harvesting complex of photosystem II (LHCII) is the main contributor to sunlight energy harvesting in plants. The flexible design of LHCII underlies a photoprotective mechanism whereby this complex switches to a dissipative state in response to high light stress, allowing the rapid dissipation of excess excitation energy (NPQ). In this work, we stabilized the quenched conformation of single LHCII trimers after immobilization of the complexes in polyacrylamide gels to impede protein interactions. A comparison of their pigment excited-state dynamics with quenched LHCII aggregates in buffer revealed the presence of a new spectral band at 515 nm arising after chlorophyll excitation. This is suggested to be the signature of a carotenoid excited state, linked to the quenching of chlorophyll singlet excited states. Our data highlight the marked sensitivity of pigment excited-state dynamics in LHCII to structural changes induced by the environment.