Kinetics and structure during self-assembly of oppositely charged proteins in aqueous solution

Self-assembly in aqueous solution of two oppositely charged globular proteins, hen egg white lysozyme (LYS) and bovine calcium-depleted α-lactalbumin (apo α-LA), was investigated at pH 7.5. The aggregation rate of equimolar mixtures of the two proteins was determined using static and dynamic light scattering as a function of the ionic strength (15-70 mM) and protein concentration (0.28-2.8 g/L) at 25 and 45 °C. The morphology of formed supramolecular structures was observed by confocal laser scanning microscopy. When the two proteins are mixed, small aggregates were formed rapidly that subsequently grew by collision and fusion. The aggregation process led on larger length scales to irregularly shaped flocs at 25 °C, but to monodisperse homogeneous spheres at 45 °C. Both the initial rate of aggregation and the fraction of proteins that associated decreased strongly with decreasing protein concentration or increasing ionic strength but was independent of the temperature.     

Control of nucleation in microtubule self-assembly

The inhibition of the rate and amplitude of assembly of microtubule protein at low GTP concentration is shown by measurement of microtubule length distributions to be due to the suppression of microtubule nucleation. This inhibitory effect is enhanced by GDP added before assembly, but can be overcome by a number of molecules such as pyrophosphate or ADP. The selective inhibition of nucleation by GDP in vitro, which occurs in addition to inhibition of elongation, could provide a mechanism for the control of spontaneous microtubule nucleation in vivo.     

Spontaneous self-assembly of SC3 hydrophobins into nanorods in aqueous solution

Hydrophobins are small surface active proteins secreted by filamentous fungi. Because of their ability to self-assemble at hydrophilic-hydrophobic interfaces, hydrophobins play a key role in fungal growth and development. In the present work, the organization in aqueous solution of SC3 hydrophobins from the fungus Schizophyllum commune was assessed using Dynamic Light Scattering, Atomic Force Microscopy and fluorescence spectroscopy. These complementary approaches have demonstrated that SC3 hydrophobins are able not only to spontaneously self-assemble at the air-water interface but also in pure water. AFM experiments evidenced that hydrophobins self-assemble in solution into nanorods. Fluorescence assays with thioflavin T allowed establishing that the mechanism governing SC3 hydrophobin self-assembly into nanorods involves β-sheet stacking. SC3 assembly was shown to be strongly influenced by ionic strength and solution pH. The presence of a very low ionic strength significantly favoured the protein self-assembly but a further increase of ions in solution disrupted the protein assembly. It was assessed that solution pH had a significant effect on the SC3 hydrophobins organization. In peculiar, the self-assembly process was considerably reduced at acidic pH. Our findings demonstrate that the self-assembly of SC3 hydrophobins into nanorods of well-defined length can be directly controlled in solution. Such control allows opening the way for the development of new smart self-assembled structures for targeted applications.     

Interactions between bacterial flagellar axial proteins in their monomeric state in solution

The axial structure of the bacterial flagellum is composed of many different proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymerization appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out. We therefore studied self and cross-association between hook protein, flagellin and three hook-associated proteins, HAP1, HAP2 and HAP3, in all possible pairs, by gel-filtration and analytical centrifugation, and found interactions in the following two cases only. Flagellin facilitated HAP3 aggregation into beta-amyloid-like filaments, but without stable binding between the two. Addition of HAP3 to HAP2 resulted in disassembly of preformed HAP2 decamers and formation of stable HAP2-HAP3 heterodimers. HAP2 missing either of its disordered terminal regions did not form the heterodimer, whereas HAP3 missing either of its disordered terminal regions showed stable heterodimer formation. This polarity in the heterodimer interactions suggests that the interactions between HAP2 and HAP3 in solution are basically the same as those in the flagellar axial structure. We discuss these results in relation to the assembly mechanism of the flagellum.     

Effects of four disaccharides on nucleation and growth of ice crystals in concentrated glycerol aqueous solution

Devitrification has been determined to be one of the major causes of cell death in cryopreservation by vitrification method. Reliable quantification of the nucleation and growth of ice crystals of devitrification is of great importance for the optimization of the vitrification solutions. In the present study, cryomicroscopy was used to investigate the nucleation and growth of ice crystals in concentrated glycerol aqueous solution (60 wt%) in the presence of sucrose, trehalose, maltose and lactose. Results showed that sucrose rather than trehalose seems to be the most effective one to inhibit the nucleation and ice growth, despite the excellent inhibitory ability of trehalose on ice growth that has been confirmed in many researches. Hence, for ice inhibition, sucrose was a more effective disaccharide additive to suppress nucleation and growth of ice crystals that occurred during devitrification in concentrated glycerol solutions.     

A C-RING-like domain participates in protein self-assembly and mineral nucleation

AP7 is a nacre-associated protein of the mollusk shell that forms supramolecular assemblies that nucleate single-crystal aragonite in vitro. AP7 possesses two major sequence regions: a random coil 30-amino acid N-terminal domain (AP7N) and a partially disordered 36-amino acid C-terminal domain (AP7C) that exhibits imperfect sequence homology to the C subclass of the intracellular RING domain family. We report here new findings that implicate the C-RING domain in AP7-mediated supramolecular assembly and single-crystal mineral formation. AP7 protein spontaneously self-assembles over a pH range of 4-9 and is monomeric at pH >9.5. AP7N and AP7C both oligomerize over the pH range of 4-9, with the AP7C sequence closely resembling AP7 in terms of particle morphology and size. In vitro mineralization experiments demonstrate that both AP7N and AP7C form supramolecular assemblies that nucleate single-crystal calcium carbonates. Comparison of previously published nuclear magnetic resonance-based structures of AP7C and AP7N reveals the significant presence of complementary anionic-cationic electrostatic molecular surfaces on AP7C that are not found on AP7N, and this may explain the noted discrepancies between the two domains in terms of self-assembly and single-crystal nucleation. We conclude that the C-RING-like sequence is an important site for AP7 self-association and mineral nucleation, and this represents the first known instance of a RING-like sequence performing these functions within an extracellular protein.     

The solution structures and activity of caerin 1.1 and caerin 1.4 in aqueous trifluoroethanol and dodecylphosphocholine micelles

The caerin 1 peptides are among the most powerful of the broad-spectrum antibiotic amphibian peptides. Caerin 1.1 has previously been shown to form an amphipathic helix-bend-helix structure in aqueous trifluoroethanol (H. Wong, J. H. Bowie, and J. A. Carver European Journal Biochemistry, 1997, Vol. 247, pp. 545-557) and structure-activity relationship studies indicate that both helices are required for activity, as well as flexibility in the bend region connecting the two. The structure of caerin 1.1 in dodecylphosphocholine micelles was investigated and shown to be very similar to that determined in aqueous trifluoroethanol. Caerin 1.4, which is identical to caerin 1.1, but with serine residues replacing Val5 and Gly7, is less active than caerin 1.1 against most bacterial species but has improved activity against Escherichia coli and Micrococcus luteus. The solution NMR structure of caerin 1.4 was determined in both aqueous trifluoroethanol and dodecylphosphocholine micelles, and was shown to be similar to caerin 1.1. It was concluded that differences in the hydrophobicity and hydrophilic angle of the first helix are probably responsible for the different spectra of antibacterial activity. The similarity of the structures calculated in aqueous trifluoroethanol and dodecylphosphocholine micelles suggests that, for caerin 1.1 and 1.4, these solvent systems are equally as good at representing a membrane environment.     

Simulation of self-assembly behaviour of fluorinated phospholipid molecules in aqueous solution by dissipative particle dynamics method

In order to prepare novel biomaterials, it is essential to investigate the self-assembly behaviour of molecules containing both hydrophobic and hydrophilic groups, and to understand their structural change and morphological development. In this paper, we studied the self-assembly behaviour of fluorinated double-chain phospholipid molecules in aqueous solution at various simulation steps, concentrations, temperatures and pH values via the dissipative particle dynamics simulation method. The self-assembly behaviours of hydrogenated analogues and fluorinated single-chain phospholipids at various concentrations were also investigated for comparison. It was found that all molecules could form microsphere at low concentration, and aggregated to form various shapes with the increase of concentration. Fluorinated double-chain phospholipids were apt to form bilayer membrane more easily than hydrogenated/fluorinated single-chain phospholipids. Besides concentration, temperature and pH value of the aqueous solution also influence the self-assembly behaviour of the investigated molecules. A stable bilayer membrane could be achieved for the fluorinated double-chain phospholipids at a relatively high concentration when pH value and temperature of aqueous solution were close to physiological conditions, i.e., pH 7 and T = 37°C. This work provides a direct ‘observation’ of self-assembly behaviour in the molecular level, which is important for the development of novel biomaterials, where surface structure is required to be well controlled.     

The structures of solvated methylmercury(II) chlolade,bromide and iodide in pyridine solution; Implications for aqueous solution

The structures of solvated methylmercury(II) halides in pyridine solution were determined by a large angle X-ray scattering technique. Near-linear CH₃HgX (X = Cl, Br and I) species solvated by two weakly-coordinated pyridine molecules are indirectly interpreted. Additional mercury-pyridine interactions, through van der Waals forces, are found at the sum of the van der Waals radii. The HgX bond distances in the methylmercury(II) halides are found to be 2.325(8), 2.480(3) and 2.649(3) Å for chloride, bromide and iodide, respectively. The HgC bond distances are assumed to be ∼2.08 Å. This interaction is indicated in the radial distribution functions. The bond distance between mercury and the two solvating pyridine molecules is ∼2.8 Å, e.g., 2.84(2) Å in methylmercury(II) bromide. The additional mercury interactions with roughly two pyridine molecules at the sum of van der Waals radii are revealed at around 3.15 Å. Comparison between Raman stretching vibrations and the solvated structures of methylmercury(II) complexes found in various solvents indicates a lower limit in solvent donor property for the formation of solvate bonds to mercury for the methylmercury(II) halides.     

Synthesis of amino-analogs of bacteriochlorophyll-d and their self-aggregation in an aqueous micelle solution

Zinc methyl 3-aminomethyl- and 3-(1-aminoethyl)-pyropheophorbides-a were prepared by modifying naturally occurring chlorophyll-a. The synthetic amino-analogs of bacteriochlorophyll-d self-aggregated in an aqueous micelle solution to give large oligomers with red-shifted and broadened electronic absorption bands. The spectra of these self-aggregates were similar to those of bacteriochlorophyll self-aggregates in the main light-harvesting antennas of green photosynthetic bacteria. The 3¹-amino groups were alternative to the 3¹-hydroxy groups in natural bacteriochlorophylls-c/d/e/f.     

The structure of gellan in dilute aqueous solution

A full assignment of high-field nmr spectra of gellan was obtained in dilute aqueous solution by performing a series of selective one-dimensional nmr experiments. The observed nuclear Overhauser effects (NOEs) cannot be interpreted assuming that each sugar residue is intrinsically rigid and in a chair conformation. In fact, the rhamnose residue gives strong NOE contacts coherent only with an equilibrium involving both a chair as well as a boat (or a hemiboat) conformation. Molecular dynamic calculations performed on a heptamer with a central rhamnose support the above finding, and show a structure based on a very stiff single chain in which it is present a flipping of the rhamnose residue. At low temperatures (5-20 degrees C) in very dilute solutions (0.018 mg/mL) nmr spectra show a splitting of the resonance due to the methyl group of rhamnose residue, thus confirming the presence of a slow equilibrium among different conformers.     

Solution structures of ferrihaem in some dipolar aprotic solvents and their binary aqueous mixtures

1. Conductivity and u.v. and visible spectroscopic techniques were used to investigate the solution structure of the prosthetic group of the ferric haemoproteins (ferrihaem) in dimethyl sulphoxide, NN-dimethylacetamide, NN-dimethylformamide and sulpholane, and certain of their aqueous mixtures. 2. In neutral or acid dimethyl sulphoxide, chlorohaemin is monomeric and completely dissociated into Cl⁻ion and a ferrihaem species with dimethyl sulphoxide molecules in the fifth and sixth co-ordination positions on iron. 3. In neutral NN-dimethylacetamide and NN-dimethylformamide chlorohaemin is monomeric but is largely undissociated, giving different spectra from that of chlorohaemin in dimethyl sulphoxide. On acidification, dissociation occurs and the dimethyl sulphoxide type of spectrum results. 4. Studies in a fourth solvent, sulpholane, indicate that solvent co-ordinating power (ligand strength) rather than bulk dielectric constant is responsible for dissociation of chlorohaemin. 5. In neutral dimethyl sulphoxide–water mixtures chlorohaemin remains monomeric and completely dissociated, and spectra are independent of mixture composition, except at high water concentrations, when precipitation occurs. In alkaline dimethyl sulphoxide–water mixtures, where the complete solvent mixture range is accessible, ferrihaem is polymeric (probably dimeric) and spectra are dependent on solvent composition. A quantitative analysis indicates that the spectral changes are due to replacement by water of one molecule of co-ordinated dimethyl sulphoxide per ferrihaem aggregate, and do not involve a two-molecule replacement as has been suggested for the alkaline pyridine–water system.     

Porphyrins affect the self-assembly of tubulin in solution

Self-assembly of tubulin heterodimers in solution has been studied in the past to predict the effects that ligands and/or conformational changes have on the formation of tubulin filaments. Self-assembly of tubulin in solution has produced formations similar to cellular microtubules (MTs). The present study reports on the effects that two porphyrins (protoporphyrin IX, PPIX and tetrakis(4-sulfonatophenyl)porphyrin, TPPS) produce on the self-assembly of tubulin α,β-heterodimers in buffer solution. The study shows that, when incubated simultaneously with MT-stabilizing ligands (i.e., paclitaxel and guanosine triphosphate, GTP), porphyrins do not affect the ability of tubulin to form MT. However, if paclitaxel and GTP are added after tubulin has been allowed to self-assemble in the presence of either porphyrin, the ability to form MT-like structures is reduced or suppressed. We suggest that this effect is due to the formation of porphyrin-mediated aggregates that cannot be broken or elongated by the addition of GTP or paclitaxel.     

Ice nucleation and supercooling behavior of polymer aqueous solutions

We determined the homogeneous nucleation temperature depression, ΔT_f,hom, the equilibrium melting point depression, ΔT_m, and the value λ, which can be obtained from the linear relationship ΔT_f,hom = λΔT_m, for aqueous solutions of PEG (200-20,000 g mol⁻¹), PVP (10,000, 35,000, 40,000 g mol⁻¹), and dextran (10,000 g mol⁻¹) in the concentration range 0-40 wt% using the emulsion method. The molecular weight dependence of T_f,hom, T_m, and λ in PEG aqueous solutions was found to change in the vicinity of Mw 600-1540 at all concentrations. In addition, it was confirmed that for all of the polymers studied, there was a good linear relationship between λ and the logarithmic value of the self-diffusion coefficient D₀ of the solute molecule. These results indicate that the parameters that describe non-equilibrium freezing, such as T_f,hom and λ, are dependent on solution properties such as viscosity and self-diffusion of solute molecules.     

The behavior of inososes in neutral and basic aqueous solution

N.m.r. spectra (¹H and ¹³C) have shown that, of three inososes studied, the 2,3,4,6/5-isomer exists in solution as the keto form, and the 2,4,6/3,5-isomer is partially, and the 2,3,5/4,6-isomer is almost fully, hydrated. In alkaline solution, each of the inososes rapidly loses a molecule of water, to give trans-2,3,4,5-tetrahydroxy-2-cyclohexen-1-one. On acetylation in the presence of a base, this compound gives tetraacetoxybenzenes; hydrogenation yields several cyclohexanepentols.     

Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans.     

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.