Enhanced stability of heterologous proteins by supramolecular self-assembly

Recently, we reported on the dual function of human ferritin heavy chain (hFTN-H) used for the fusion expression and solubility enhancement of various heterologous proteins: (1) high-affinity interaction with HSP70 chaperone DnaK and (2) formation of self-assembled supramolecules with limited and constant sizes. Especially the latter, the self-assembly function of hFTN-H is highly useful in avoiding the undesirable formation of insoluble macroaggregates of heterologous proteins in bacterial cytoplasm. In this study, using enhanced green fluorescent protein (eGFP) and several deletion mutants of Mycoplasma arginine deiminase (ADI_132–410) as reporter proteins, we confirmed through TEM image analysis that the recombinant fusion proteins (hFTN-H::eGFP and hFTN-H::ADI_132–410) formed intracellular spherical particles with nanoscale diameter (≈10 nm), i.e., noncovalently cross-linked supramolecules. Surprisingly, the supramolecular eGFP and ADI showed much enhanced stability in bioactivity. That is, the activity level was much more stably maintained for the prolonged period of time even at high temperature, at high concentration of Gdn–HCl, and in wide range of pH. The stability enhancement by supramolecular self-assembly may make it possible to utilize the protein supramolecules as novel means for drug delivery, enzymatic material conversion (biotransformation), protein chip/sensor, etc. where the maintenance of protein/enzyme stability is strictly required. Jin-Seung Park and Ji-Young Ahn contributed equally to this work.     

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.     

Supramolecular complexes for nanomedicine

Host-guest interactions studied in supramolecular chemistry have been inspired by interactions between enzymes and substrates. Furthermore, most of the interactions involved in the cells are based on non-covalent bonds between two or more molecules. The common aspects between supramolecular chemistry and medicine have led to the development of a “new” area called “supramolecular medicine”, in which non-covalent interactions and self-assembly processes are applied within several medical fields.The object of this Digest is to offer an account of how some macrocyclic hosts (e.g. cucurbiturils, cyclodextrins, pillararenes and calixarenes) are employed in supramolecular medicine creating new supramolecular hydrogels used as biomaterials for human tissue in regenerative medicine, and a diagnostic instrument, in-vitro and in-vivo, for the detection of diseases, as well as for the investigation of cell morphology.     

Supramolecular platinum complexes for cancer therapy

The rise of supramolecular chemistry offers new tools to design therapeutics and delivery platforms for biomedical applications. This review aims to highlight the recent developments that harness host-guest interactions and self-assembly to design novel supramolecular Pt complexes as anticancer agents and drug delivery systems. These complexes range from small host-guest structures to large metallosupramolecules and nanoparticles. These supramolecular complexes integrate the biological properties of Pt compounds and novel supramolecular structures, which inspires new designs of anticancer approaches that overcome problems in conventional Pt drugs. Based on the differences in Pt cores and supramolecular structures, this review focuses on five different types of supramolecular Pt complexes, and they include host-guest complexes of the FDA-approved Pt(II) drugs, supramolecular complexes of nonclassical Pt(II) metallodrugs, supramolecular complexes of fatty acid-like Pt(IV) prodrugs, self-assembled nanotherapeutics of Pt(IV) prodrugs, and self-assembled Pt-based metallosupramolecules.     

G-quartet self-assembly under osmotic pressure: remote control by vesicle shrinking rather than stress

Does osmotic pressure stimulate assembly or disassembly of supramolecules in vesicles? Self‐assembly was conceivable as intravesicular response to osmotic shrinking upon application of extravesicular overpressure, whereas disassembly was conceivable as a response to bilayer stress in hyperosmotic vesicles. Self‐assembly of guanosine 5′‐monophosphates (GMPs) into G‐quartets was selected to investigate the nature of remote control of supramolecular chemistry within vesicles by osmotic pressure. Using circular dichroism spectroscopy to selectively detect G‐quartets, we found that extravesicular overpressure stimulates intravesicular self‐assembly, whereas underpressure stimulates disassembly. G‐quartet self‐assembly by osmotic pressure exhibited ion‐selective metal‐cation templation, as expected. The key conclusions are that supramolecular chemistry within vesicles is governed by vesicle shape rather than vesicle stress and that detection of osmotic pressure by CD spectroscopy is an interesting alternative to the commonly used methods based on fluorescence self‐quenching. Chirality 15:766–771, 2003. © 2003 Wiley‐Liss, Inc.     

Equilibrium ligand binding assays using labeled substrates: nature of the errors introduced by radiochemical impurities

The effects of radiochemical impurities in a labeled substrate on the characteristics of the experimental equilibrium binding plots were examined. The protein (receptor) was assumed to be a monomer or an oligomer composed of identical, noninteracting subunits. The substrate was assumed to be chemically and radiochemically impure (Case I) or just radiochemically impure (Case II). In both cases, the apparent free substrate concentration required for half-saturation of the protein, [S]_0.5,app, increases linearly with increasing total protein concentration. Reciprocal plots and Scatchard plots are nonlinear. The curvature of both plots is opposite to that observed for the heterogeneity of binding sites. Hill plots are curved with average slopes >1 in the region of half-saturation. If the radiochemical impurity goes undetected, the experimental data might lead an investigator to suggest a number of unnecessarily complicated binding models. The plots obtained in the presence of a radiochemical impurity are very similar to those seen when the receptor protein is a dissociable dimer and K_s (monomer) <K_s (dimer). However, in the dimer model the variation of [S]_0.5 with total protein concentration is nonlinear. The most direct way of assessing the radiochemical purity of a labeled substrate is to vary the binding protein concentration at a fixed concentration of S¹. If all of the radioactivity resides in S¹, the concentration of the PS¹ complex will approach [S¹]_t as [P]_t increases, while the free unbound radioactivity will be driven toward zero. If the labeled substrate is radiochemically impure, “saturating” protein will not bind all of the label. This procedure will detect some types of major impurities missed by paper chromatography (e.g., ³H₂O and nonreactive isomers of S¹).     

Synthetic Approach to biomolecular science by cyborg supramolecular chemistry

Background

To imitate the essence of living systems via synthetic chemistry approaches has been attempted. With the progress in supramolecular chemistry, it has become possible to synthesize molecules of a size and complexity close to those of biomacromolecules. Recently, the combination of precisely designed supramolecules with biomolecules has generated structural platforms for designing and creating unique molecular systems. Bridging between synthetic chemistry and biomolecular science is also developing methodologies for the creation of artificial cellular systems.

20 Supramolecular polymerization of nucleobase-like monomers in water

Elucidating the physiochemical principles that govern molecular self-assembly is of great importance for understanding biological systems and may provide insight into the emergence of the earliest macromolecules of life, an important challenge facing the RNA World hypothesis. Self-assembly results from a delicate balance between multiple noncovalent interactions and solvent effects, but achieving efficient self-assembly in aqueous solution with synthetic molecules has proven particularly challenging. Here, we demonstrate how two physical properties – monomer solubility and large hydrophobic surfaces of intermediate structures – are key elements to achieving supramolecular polymers in aqueous solution (Cafferty et al., 2013). Applying these two principles, we report the highly cooperative self-assembly of two weakly interacting, low molecular weight monomers [cyanuric acid and a modified triaminopyrimidine] into a water-soluble supramolecular assembly (see scheme below). The observed equilibrium between only two appreciably populated states – free monomers and supramolecular assemblies – is in excellent agreement with the values previously determined for the free energy of hydrogen bonding (Klostermeier & Millar, 2002), π???π stacking (Frier et al., 1985), and the calculated free energy penalty for the solvation of hydrophobic structures in water (Chandler, 2005). The similarity of the molecules used in this study for the nucleobases found in contemporary nucleic acids and the demonstration that these monomers assemble while the natural nucleobases do not, suggests that the first informational polymers may have emerged from a similar self-assembly process, if the nucleobases were different then they are today (Hud et al., 2013).     

Self-assembly of DNA-polymer complexes using template polymerization.

The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles.     

Cooperative effects of CTP on calf liver CTP synthetase.

In all previous kinetics studies of calf liver CTP synthetase, simple Michaelis-Menten hyperbolic plots were obtained. In this study it was shown that calf liver CTP synthetase could generate sigmoidal kinetic plots as a function of the substrate UTP when in the presence of the product of the reaction, CTP. The Hill number was estimated to be 2.8. The enzyme did not generate sigmoidal plots as a function of the other substrates (L-glutamine and ATP) either in the presence or absence of CTP. Thus, CTP apparently induced changes in the liver enzyme which altered the binding of UTP to the enzyme by acting at a site distinct from the UTP binding site (allosteric site). This concept was further strengthened by the fact that 3-deazaUTP, a known competitive inhibitor of the liver enzyme, did not induce sigmoidal kinetic plots. It was also shown that CTP had no effect upon the dimerization of the enzyme, thus ruling out monomer to dimer transitions as a potential mechanism for the observed sigmoidal kinetics.     

The surface location of individual residues in a bacterial S-layer protein

Bacterial surface layer (S-layer) proteins self-assemble into large two-dimensional crystalline lattices that form the outermost cell-wall component of all archaea and many eubacteria. Despite being a large class of self-assembling proteins, little is known about their molecular architecture. We investigated the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 to identify residues located at the subunit-subunit interface and to determine the S-layer's topology. Twenty-three single cysteine mutants, which were previously mapped to the surface of the SbsB monomer, were subjected to a cross-linking screen using the photoactivatable, sulfhydryl-reactive reagent N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide. Gel electrophoretic analysis on the formation of cross-linked dimers indicated that 8 out of the 23 residues were located at the interface. In combination with surface accessibility data for the assembled protein, 10 residues were assigned to positions at the inner, cell-wall-facing lattice surface, while 5 residues were mapped to the outer, ambient-exposed lattice surface. In addition, the cross-linking screen identified six positions of intramolecular cross-linking within the assembled protein but not in the monomeric S-layer protein. Most likely, these intramolecular cross-links result from conformational changes upon self-assembly. The results are an important step toward the further structural elucidation of the S-layer protein via, for example, X-ray crystallography and cryo-electron microscopy. Our approach of identifying the surface location of residues is relevant to other planar supramolecular protein assemblies.     

Layer-by-layer self-assembly of supramolecular and biomolecular films

In this paper, we give a short account on recent studies of layer-by-layer self-assembly of supramolecular and biomolecular films. Such films are built up from layers of macro-ions with opposing charge. A simple film can be obtained by alternating the adsorption of two components: a flexible, synthetic polycation chains and a supramolecular or biomolecular moiety. We focus on three examples, in which the second component consists either of a supramolecular metal-organic complex (MOC), a nucleic acid, or a biological membrane patch (purple membrane). While the flexible polvcation chains (as well as eventual annealing layers) ensure a uniform build-up of the chain, the second macromolecular component may be used to functionalize the films. The combination of layer-by-layer self-assembly and biotechnologically relevant macromolecules may lead to new devices or biomaterial applications. To this end, precise studies of the deposition process and the film structure are needed. Here, we focus on interface sensitive scattering techniques for the structural analysis.     

Supramolecular photochemical self-assemblies for fluorescence "turn on" and "turn off" assays for chem-bio-helices.

We describe the development of an optical sensing system for the high-throughput screening (HTS) of a broad range of biological molecules, whole cells, organisms and pathogens, and illustrate the technology applications by a hyaluronidase enzyme activity assay as a specific example. At the core of the technology described in this paper, is the exciton concept that is relevant to molecular aggregation. J-aggregates of cyanine dyes have a narrower, red-shifted absorption band compared to monomer. We demonstrate that self-assembly may be driven by the helicogenic nature of the cyanine dye, converting the linear polymers of hyaluronic acid or carboxymethyl cellulose into supramolecular helical assemblies. This self-assembly is accompanied by an intense, sharp, red-shifted J-aggregate fluorescence. We utilized this property to develop an assay for the enzyme hyaluronidase, based upon the concept of "scaffold destruction," whereby the disruption/destruction of the hyaluronic acid polymer by hyaluronidase is accompanied by an attenuation of light emission from the J-aggregate. The extent of light attenuation provides an index of hyaluronidase activity. Other polymers of carbohydrates, proteins, nucleic acids and chemical polymers (such as the carbon nanotube) might provide a similar scaffold for helicogenic dyes upon which molecular aggregation can occur. A key feature of these assays is that they are label-free.     

Interaction of oligomycin with the pathway of mitochondrial energy transfer

In bovine heart submitochondrial particles, energy-linked reactions driven by coupling to aerobic oxidation, are stimulated by the antibiotic oligomycin. Hill plots of the stimulation show two distinct slopes, indicating that oligomycin interferes with two steps in the energy conservation process. ATP-driven energy-linked reactions are stimulated at low concentrations of oligomycin and inhibited at high concentrations. The Hill plots for the stimulation as well as the inhibition show only one slope. The addition of energy coupling factors does not alter the slope of the Hill plots. In intact mitochondria, the inhibition of ATP-P_i exchange and active oxidation by oligomycin yield single-slope Hill plots, while the inhibition of dinitrophenol-stimulated ATPase yields a Hill plot with two distinct slopes. The results of Hill analysis are interpreted on the basis of the mechanism of oligomycin action proposed by Lee and Ernster.     

HPRT and the Lesch—Nyhan syndrome

The view is presented that extracellular architecture in plant cell walls results from an interplay between molecular self-assembly and mechanical reorientation due to growth forces. A key initial self-assembly step may involve hemicelluloses. It is suggested that hemicelluloses may self-assemble into a helicoid via a cholesteric liquid crystalline phase; the detailed molecular structure of hemicelluloses (stiff backbone, bulky side chains, and the presence of asymmetric carbon atoms) is shown to be consistent with cholesteric requirements for such self-assembly. Since hemicelluloses are hydrogen-bonded to the periphery of cellulose microfibrils, the cellulose could then itself become helicoidally arranged. Such ‘universal plywood’ structure is found in the walls of a wide variety of plants, and in several types of cell (including wood). The permanent effects of growth stresses on patterns seen in sections of helicoids are displayed by computer graphics plots, and the expected changes in stiffness are calculated.     

Self-association of haemoglobin Olympia (alpha 2 beta 2 20 (B2) Val----Met). A human haemoglobin bearing a substitution at the surface of the molecule

Oxygenation measurements at equilibrium were carried out for solutions of pure haemoglobin (Hb) Olympia (alpha 2 beta 2 20 (B2) Val----Met) at 200 microM (haem) and revealed a high oxygen affinity (P50 = 4.2 torr at pH 7.20, 25 degrees C) compared to HbA (P50 = 5.6 torr), with the Hill coefficient (eta H) reduced from the normal value of 2.9 to 2.5 for Hb Olympia at neutral pH. 2,3-Diphosphoglycerate and chloride effects were normal, but measurements of the alkaline Bohr effect indicated an excess Bohr effect of about 20% for Hb Olympia. Precise determinations of the oxygen binding curves gave the unexpected finding of a dependence of co-operativity on pH with eta H rising from 2.4 at pH 6.8 to 3.0 at pH 8. Moreover, the Hill coefficient was dependent upon the concentration at alkaline pH and fell to 1.8 in low concentration solutions (approximately 30 microM-haem) of the haemoglobin variant; at this concentration the Bohr effect was normal. This effect of concentration on co-operativity could be accounted for fully by the allosteric model, with introduction of Hb Olympia self-association. In this case the allosteric constant L' for the supramolecular species has the value of the allosteric constant L for the tetramer species, raised to a power equal to the number of molecules in the aggregates and modulated by the ratio of the dissociation constants of the aggregates, DNR/DNT. Model curves with N tetramers per aggregate (where N approximately 2 at pH 7.5 and N approximately 4 at pH 8.0) fully represented the concentration dependence for Hb Olympia of the eta H values and the detailed shape of the experimental curves for eta H as a function of log[y/(1-y)], the first derivative of the Hill plot. These curves are much steeper when supramolecular species are present. Direct measurements of the protein aggregation by centrifugation confirmed the presence of aggregates in the solutions of Hb Olympia. Hb Olympia is therefore one of the few examples of mutant human haemoglobins that self-associate with functional consequences in terms of oxygen binding properties.(ABSTRACT TRUNCATED AT 400 WORDS)     

HIV Rev self-assembly is linked to a molten-globule to compact structural transition

By regulating the differential expression of proviral pre mRNA in the host cell, Rev plays a crucial role in the HIV-1 life cycle. The capacity of Rev to function is intimately linked to its ability to self-associate. Nevertheless, little is known about the exact role of self-association in the molecular mechanism defining its biological activity. A prerequisite knowledge is a definition of the molecular events undertaken by Rev during the process of self-assembly. Thus, this study was initiated to monitor the structure of Rev as a function of protein concentration. Rev undergoes a structural transition as a consequence of self-assembly. This structural transition was monitored by three spectroscopic methods. The accessibility of the single tryptophan in Rev monomer to acrylamide quenching increases with decreasing protein concentration. At very low concentration of Rev, the tryptophan accessibility is close to that of an unfolded Rev. As evaluated by circular dichroism, the secondary structure of Rev is protein concentration dependent as evidenced by an increase in the magnitude of ellipticity with increasing protein concentration. Further, results from ANS binding studies indicate that the ANS binding sites in Rev experience an apparent increase in hydrophobicity as the Rev concentration was increased. These concentration dependent changes seem to reach a maximum above 5 microM Rev monomer concentration. In order to define the mode of Rev self-association sedimentation velocity and equilibrium experiments were conducted. There are evidently two consecutive progressive association processes. At protein concentrations below 0.5 mg/ml, the data from sedimentation studies can be fitted to a single isodesmic model. Simulation of velocity sedimentation profile indicates that free Rev monomer that has not entered into the association processes can best be described to exhibit a value of S(20,w) that is substantially smaller than 1.4 S, a value needed to fit the rest of the data. The latter value is consistent for a Rev monomer with the expected molecules weight and if it were to assume a compact globular shape. These spectroscopic and hydrodynamic results imply that monomeric Rev is in a molten globule state, which becomes more compact upon self-association.     

Kinetic co-operativity of monomeric mnemonical enzymes. The significance of the kinetic Hill coefficient

The expression of the kinetic Hill coefficient for a two-substrate, two-product mnemonical enzyme has been derived. Its relation with the gamma coefficient, that is the slope of the reciprocal plots for 1/[A]----O, has been established. The variation of this Hill coefficient, as a function of the second substrate and product concentrations, has been studied theoretically. Whereas the gamma coefficient does not vary as a function of the substrate and first product concentrations, the kinetic Hill coefficient does. If the enzyme is positively co-operative, the Hill coefficient increases upon increasing the second substrate concentration and decreases if the first product concentration is increased. The converse is expected to occur if the enzyme displays a negative co-operativity. The last product may either reverse a positive co-operativity into a negative one or, alternatively, strengthen an already negative co-operativity. The co-operativity generated by the mnemonical model has been compared to the kinetic behaviour of a random model. These two models have been shown to be discriminated on the basis of the departure they show with respect to the Michaelis-Menten behaviour. These theoretical considerations have been applied to previously published data, obtained with wheat germ hexokinase LI. This monomeric enzyme has a negative co-operativity with respect to the preferred substrate, glucose. The Hill coefficient decreases with MgATP concentration, increases with MgADP concentration and decreases with glucose-6-phosphate concentration. This is exactly what is to be expected on the basis of the above theory of kinetic co-operativity.     

A beta fibrillogenesis: kinetic parameters for fibril formation from congo red binding

Using Scatchard analysis, we have formulated as a function of time and pH the relationship between the binding of Congo red to Alzheimer's beta-amyloid and the aggregation number (i.e., monomer concentration within fibrils) as defined by nucleation-dependent self-assembly. This provides a basis on which to determine the kinetic parameters for fibril formation from the observed concentration of bound Congo red.     

Dissociation of the GroEL-GroES asymmetric complex is accelerated by increased cooperativity in ATP binding to the GroEL ring distal to GroES

A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance. Excellent fits of the data were obtained using a double-exponential equation with a linear drift. Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP. The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES. Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring. It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex.