Single-chain Complex Research Articles

Directly visualizing individual polyorganophosphazenes and their single-chain complexes with proteins

Polyorganophosphazenes are water-soluble macromolecules with immunoadjuvant activity that self-assemble with proteins to enable biological functionality. Direct imaging by cryogenic electron microscopy uncovers the coil structure of those highly charged macromolecules. Here, we successfully visualize individual polymer chains within the vitrified state in the absence of additives for contrast enhancement which is attributed to the high mass contrast of the inorganic backbone. Upon assembly with proteins, multiple protein copies bind at the single polymer chain level resulting in structures reminiscent of compact spherical complexes or stiffened coils. The outcome depends on protein characteristics and cannot be deduced by commonly used characterization techniques, such as light scattering, thus revealing direct morphological insights crucial for understanding biological activity. Atomic force microscopy supports the morphology outcomes while advanced analytical techniques confirm protein-polymer binding. The chain visualization methodology provides tools for gaining insights into the processes of supramolecular assembly and mechanistic aspects of polymer-enabled vaccine delivery.

Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules.

Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (β2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.

Molecular Characterization of the Native (Non-Linked) CD160–HVEM Protein Complex Revealed by Initial Crystallographic Analysis

An increasing number of surface-exposed ligands and receptors acting on immune cells are being considered as a starting point in drug development applications. As they are dedicated to manipulating a wide range of immune responses, accurately predicting their molecular interactions will be necessary for the development of safe and effective therapeutics to enhance immune responses and vaccination. Here, we focused on the characterization of human CD160 and HVEM immune receptors, whose mutual engagement leads to bidirectional signaling (e.g., T cell inhibition, natural killer cell activation or mucosal immunity). In particular, our study reports on the molecule preparation, characterization and initial crystallographic analysis of the CD160–HVEM complex and both HVEM and CD160 in the absence of their binding partner. Despite the importance of the CD160–HVEM immune signaling and its therapeutic relevance, the structural and mechanistic basis underlying CD160–HVEM engagement has some controversial evidence. On one hand, there are studies reporting on the CD160 molecule in monomeric form that was produced by refolding from bacterial cells, or as a covalently linked single-chain complex with its ligand HVEM in insect cells. On the other hand, there are older reports providing evidence on the multimeric form of CD160 that acts directly on immune cells. In our study, the native non-linked CD160–HVEM complex was co-expressed in the baculovirus insect host, purified to homogeneity by anion-exchange chromatography to provide missing evidence of the trimeric form in solution. Its trimeric existence was also confirmed by the initial crystallographic analysis. The native CD160–HVEM complex crystallized in the orthorhombic space group with unit cell parameters that could accommodate one trimeric complex (3:3) in an asymmetric unit, thus providing ample space for the multimeric form. Crystals of the CD160–HVEM complex, CD160 trimer and HVEM monomer (reported in two space groups) diffracted to a minimum Bragg spacing of 2.8, 3.1 and 1.9/2.1 Å resolution, respectively. The obtained data will lead to elucidating the native structure of the complex.

Viscoelasticity and Solution Stability of Cyanoethylcellulose with Different Molecular Weights in Aqueous Solution.

Water-soluble cellulose ethers are widely used as stabilizers, thickeners, and viscosity modifiers in many industries. Understanding rheological behavior of the polymers is of great significance to the effective control of their applications. In this work, a series of cyanoethylcellulose (CEC) samples with different molecular weights were prepared with cellulose and acrylonitrile in NaOH/urea aqueous solution under the homogeneous reaction. The rheological properties of water-soluble CECs as a function of concentration and molecular weight were investigated using shear viscosity and dynamic rheological measurements. Viscoelastic behaviors have been successfully described by the Carreau model, the Ostwald-de-Waele equation, and the Cox–Merz rule. The entanglement concentrations were determined to be 0.6, 0.85, and 1.5 wt% for CEC-11, CEC-7, and CEC-3, respectively. All of the solutions exhibited viscous behavior rather than a clear sol-gel transition in all tested concentrations. The heterogeneous nature of CEC in an aqueous solution was determined from the Cox–Merz rule due to the coexistence of single chain complexes and aggregates. In addition, the CEC aqueous solutions showed good thermal and time stability, and the transition with temperature was reversible.

MX-type single chain complexes with an aromatic in-plane ligand: incorporation of aromatic interactions for stabilizing the chain structure.

MX-type one-dimensional complexes [PtIV(amp)2Br2][PtII/IV(amp)2Br]2(HSO4)2(SO4)2·13H2O (3) and [PtIV(amp)2Br2][PtII/IV(amp)2Br]2(H2PO4)6·8H2O (4) were synthesized as the first analogue containing only an aromatic in-plane ligand. The Pt-Br chain structures of 3 and 4 are stabilized by both the hydrogen-bond network along the chain and the π-stacking via intercalated Pt(iv) complexes. Structural and spectroscopic studies indicated that both 3 and 4 form the Pt(ii/iv) mixed valence state.

Simulation study on the effects of chemical structure and molecular size on the acceptor strength in poly(3-hexylthiophene)-based copolymer with alternating donor and acceptor for photovoltaic applications

This research assessed the effects of various chemical structures and molecular sizes on the simulated geometric parameters, electron structures, and spectroscopic properties of single-chain complex alternating donor–acceptor (D–A) monomers and copolymers that are intended for use as photoactive layer in a polymer solar cell by using Kohn–Sham density functional theory with B3LYP exchange-correlation functional. The 3-hexylthiophene (3HT) was selected for electron donor, while eight chemicals, namely thiazole (Z), thiadiazole (D), thienopyrazine (TP), thienothiadiazole (TD), benzothiadiazole (BT), thiadiazolothieno-pyrazine (TPD), oxadiazole (OXD) and 5-diphenyl-1,2,4-triazole (TAZ), were employed as electron acceptor functional groups. The torsional angle, bridge bond length, intramolecular charge transfer, energy levels, and molecular orbitals were analyzed. The simulation results reveal that the geometry and electron structure of donor–acceptor monomer and copolymer are significantly impacted by heterocyclic rings, heteroatoms, fused rings, degree of steric hindrance and coplanarity of the acceptor molecular structure. Planar conformation was obtained from the D copolymer, and a pseudo-planar structure with the TD copolymer. The TAZ acceptor exhibited strong steric hindrance due to its bulky structure and non-planarity of its structure. An analysis of the electron structures indicated that the degree of intramolecular electron-withdrawing capability had the rank order TAZ < Z < D < TPD < OXD < TP < BT < TD. The TD is indicated as the most effective acceptor among those that were simulated. However, the small energy gaps of TD as well as TPD copolymer indicate that these two copolymers can be used in transparent conducting materials. The copolymer based on BT acceptor exhibited good intramolecular charge transfer and absorbed at 656 nm wavelength which is close to the maximum flux of solar spectrum. Hence, the BT acceptor functional group provides a compromise in the characteristics of a donor–acceptor copolymer, useful in a polymeric candidate material for the photoactive layer in a polymer solar cell.

Novel designs of single-chain MHC I/peptide complex for the magnetosome display system.

The magnetic nanoparticles displaying the class I major histocompatibility complex (MHC I) were biologically synthesized using the magnetotactic bacterium Magnetospirillum magneticum AMB-1. Expression level and antigen peptide (HER263-71)-binding capability of the MHC I were evaluated on bacterial magnetic particles (BacMPs, also known as magnetosomes). Furthermore, the single-chain complexes of MHC I and HER263-71 were de novo designed for the magnetosome display system in order to improve the interaction between MHC I and HER263-71. Two types of the fusion arrangements were tested, and one of the complexes was estimated to fold into the correct conformation at the level of over 70%. In addition to the high folding ratio, an advantage of this system is that any refolding processes were not required even though the N-terminus of HER263-71 peptide is not free, which conventional bacterial expression systems have never demonstrated. The as-prepared single-chain MHC I/HER263-71 complex-displaying BacMPs (MHC I/HER2-BacMPs) specifically interacted with, and magnetically separated the HER263-71-induced cells, suggesting that the native T-cell receptor could recognize the engineered MHC I/HER2 complex on the BacMPs. By optimizing the magnetic sorting method, the MHC I/HER2-BacMPs developed in this study would be useful in immunotherapeutic applications.

The supramolecular interaction mediated chiral 1D cyanide-bridged metamagnet: synthesis, crystal structures and magnetic properties

Two cyanide-bridged enantiopure one-dimensional single chain complexes, [Mn((R,R)-Salcy)Fe(pcq)(CN)3]2n·1.5nDMF (1) and [Mn((S,S)-Salcy)Fe(pcq)(CN)3]2n·1.5nDMF (2), have been synthesized and structurally characterized. Systematically magnetic investigations show the antiferromagnetic coupling between the cyanide-bridged Mn(III)-Fe(III) centers and the interesting metamagnetic behavior at about 5.0 K resulted from the intermolecular π-π interaction.

Spectroscopic investigation on the interaction of some surfactant-cobalt(III) complexes with serum albumins

The interaction of HSA/BSA with single and double chain surfactant-cobalt(III) complexes, cis-[Co(phen)2(UA)Cl](ClO4)2·2H2O (1), cis-[Co(phen)2(UA)2](ClO4)3·2H2O (2), cis-[Co(en)2(UA)Cl](ClO4)2·2H2O (3), cis-[Co(en)2(UA)2](ClO4)3·2H2O (4), were investigated by steady state fluorescence, UV–vis absorption, synchronous, three dimensional fluorescence and circular dichroism spectroscopy. The results reveal that the quenching of HSA/BSA by all the four complexes takes place through static mechanism. The binding constant, binding sites and thermodymamic parameter were calculated. The results illustrate that the double chain surfactant-cobalt(III) complexes bind more strongly than the corresponding single chain complexes. The distance between donor (HSA/BSA) and acceptor (surfactant-cobalt(III) complexes) was obtained according to FRET. The results of synchronous, three dimensional and circular dichroism spectroscopy studies show that all the complexes caused considerable amount of conformational and some amount of environment changes in HSA/BSA.

A comparative study on the binding of single and double chain surfactant–cobalt(III) complexes with bovine serum albumin

The comparative binding effect of single and double aliphatic chain containing surfactant–cobalt(III) complexes cis-[Co(bpy)2(DA)2](ClO4)3⋅2H2O (1), cis-[Co(bpy)2(DA)Cl](ClO4)2⋅2H2O (2), cis-[Co(phen)2(CA)2](ClO4)3⋅2H2O (3), and cis-[Co(phen)2(CA)Cl](ClO4)2⋅2H2O (4) with bovine serum albumin (BSA) under physiological condition was analyzed by steady state, time resolved fluorescence, synchronous, three-dimensional fluorescence, UV–Visible absorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of BSA through a static mechanism. The binding constants (Kb) and the number of binding sites were calculated and binding constant values are found in the range of 104–105M−1. The results indicate that compared to single chain complex, double chain surfactant–cobalt(III) complex interacts strongly with BSA. Also the sign of thermodynamic parameters (ΔG°, ΔH°, and ΔS°) indicate that all the complexes interact with BSA through hydrophobic force. The binding distance (r) between complexes and BSA was calculated using Förster non-radiation energy transfer theory and found to be less than 7nm. The results of synchronous, three dimensional fluorescence and circular dichroism spectroscopic methods indicate that the double chain surfactant–cobalt(III) complexes changed the conformation of the protein considerably than the respective single chain surfactant–cobalt(III) complexes. Antimicrobial studies of the complexes showed good activities against pathogenic microorganisms.

228 Glycosylation Profiling of the HIV-1 gp120-CD4 Full Length Single Chain Complex

228 Glycosylation Profiling of the HIV-1 gp120-CD4 Full Length Single Chain Complex

Preparation and characterization of a single-chain calcineurin–calmodulin complex

Calcineurin (CN), a Ca 2+/calmodulin (CaM)-dependent serine/threonine protein phosphatase, is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). The activity of CNA is under the control of two functionally distinct, but structurally similar Ca 2+-regulated proteins, CaM and CNB. The crystal structure of the holoenzyme reveals that the N-terminus and C-terminus of CNB and the N-terminus of CNA each have a long arm not involved in the active site. We constructed a fusion of the genes of CaM, CNB and CNA in that order using linker primers containing six and ten codons of glycine. A single-chain CaM–CNB–CNA (CBA) complex was expressed and purified to near homogeneity. The single-chain complex was fully soluble, and had biochemical properties and kinetic parameters similar to single-chain CNB–CNA (BA) activated by CaM. It was not regulated by CaM and CNB, but was strongly stimulated by Mn 2+, Ni 2+ and Mg 2+. Intrinsic fluorescence spectroscopy of the complex showed a change in the environment of tryptophan in the presence of Ca 2+ and circular dichroism (CD) spectropolarimetry revealed an increase in alpha-helical content. Our findings suggest that fusion of CaM, CNB and CNA does not prevent the structural changes required for their functioning; in particular, CaM within the complex could still interact correctly with CN in the presence of Ca 2+.

In Vitro Reconstitution of Recognition and Activation Complexes between Interleukin-6 and gp130

Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific alpha-receptor (Ralpha) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6Ralpha/gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its Ralpha, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that IL-6/IL-Ralpha and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6Ralpha, and 1 gp130-CHR. Addition of the N-terminal (D1) Ig-like domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6Ralpha, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.

Syntheses, characterizations and structures of a novel double-chain polymeric cluster complex [WS 4Ag · NH 3C(CH 20H) 3 · H 20] n and its precursory single-chain complex [WS 4Ag · NH 3C(CH 20H) 3 · 2DMF] n

The title single chain complex 1 was obtained from reaction of ammonium tetrathiotungstate, silver nitrate and tris(hydroxy-methyl)-amino-methane in DMF, and the title double-chain complex 2 from the recrystallization of 1 in ethanol and a little amount of water. The anionic structure of 1 is a [WAgS 4] n linear chain, and that of 2 is a [WAgS 4] n double chain which can be viewed as two zigzag — SWSAg — chains bridged by μ in2-S atoms. In 2, H 2O molecules and NH 3C(CH 2OH) 3 + cations interact with each other through hydrogen bonding and form a two-dimensional network supramolecular structure. The inversion of configurations from 1 to 2 by re-self-assembly of [WAgS 4] fragments, as well as IR spectra of both complexes, is described.