Self-Assembly of Model Amphiphilic Peptides in Nonaqueous Solvents: Changing the Driving Force for Aggregation Does Not Change the Fibril Structure.

Within the homologous series of amphiphilic peptides AnK, both A8K and A10K self-assemble in water to form twisted ribbon fibrils with lengths around 100 nm. The structure of the fibrils can be described in terms of twisted ß-sheets extending in the direction of the fibrils, laminated to give a constant cross section of 4 nm by 8 nm. The finite width of the twisted ribbons can be reasonably explained within a simple thermodynamic model, considering a free energy penalty for the stretching of hydrogen bonds along the twisted ß-sheets and an interfacial free energy gain for the lamination of the hydrophobic ß-sheets. In this study, we characterize the self-assembly behavior of these peptides in nonaqueous solutions as a route to probe the role of hydrophobic interaction in fibril stabilization. Both peptides, in methanol and N,N-dimethylformamide, were found to form fibrillar aggregates with the same ß-sheet structure as in water but with slightly smaller cross-sectional sizes. However, the gel-like texture, the slow relaxation in dynamic light scattering experiments, and a correlation peak in the small-angle X-ray scattering pattern highlighted enhanced interfibril interactions in the nonaqueous solvents in the same concentration range. This could be ascribed to a higher effective volume of the aggregates because of enhanced fibril growth and length, as suggested by light scattering and cryogenic transmission electron microscopy analyses. These effects can be discussed considering how the solvent properties affect the different energetic contributions (hydrophobic, electrostatic, and hydrogen bonding) to fibril formation. In the analyzed case, the decreased hydrogen bonding propensity of the nonaqueous solvents makes the hydrogen bond formation along the fibril a key driving force for peptide assembly, whereas it represents a nonrelevant contribution in water.

Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin-Benson cycle.

In land plants and algae, the Calvin-Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central ß-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes-the clamp loop-which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.

Bile Salts: Natural Surfactants and Precursors of a Broad Family of Complex Amphiphiles.

Bile salts (BSs) are naturally occurring rigid surfactants with a steroidal skeleton and specific self-assembly and interface behaviors. Using bile salts as precursors, derivatives can be synthesized to obtain molecules with specific functionalities and amphiphilic structure. Modifications on single molecules are normally performed by substituting the least-hindered hydroxyl group on carbon C-3 of the steroidal A ring or at the end of the lateral chain. This leads to monosteroidal rigid building blocks that are often able to self-organize into 1D structures such as tubules, twisted ribbons, and fibrils with helical supramolecular packing. Tubular aggregates are of particular interest, and they are characterized by cross-section inner diameters spanning a wide range of values (3-500 nm). They can form through appealing pH- or temperature-responsive aggregation and in mixtures of bile salt derivatives to provide mixed tubules with tunable charge and size. Other derivatives can be prepared by covalently linking two or more bile salt molecules to provide complex systems such as oligomers, dendrimers, and polymeric materials. The unconventional amphiphilic molecular structure imparts specific features to BSs and derivatives that can be exploited in the formulation of capsules, drug carriers, dispersants, and templates for the synthesis of nanomaterials.

Bile acid derivative-based catanionic mixtures: versatile tools for superficial charge modulation of supramolecular lamellae and nanotubes.

Self-assembled structures formed by mixtures of cationic and anionic surfactants are interesting tools for applications requiring interactions with charged particles and molecules. Nevertheless, they present instability close to the equimolar composition and poor morphological versatility, which is generally restricted to vesicles and micelles. Against this general trend, we report on bile salt derivative based catanionic mixtures assembling in tubules and lamellae depending on the mixture composition. Electrophoretic mobility measurements prove that the composition also dictates their superficial charge, which can be tuned from negative to positive by increasing the positively charged surfactant fraction in the mixtures. The study of the catanionic aggregates was conducted by means of microscopy and spectroscopy techniques and compared to the self-assembly behaviors of the individual building blocks. This study broadens the so far small array of bile salt derivative catanionic systems, confirming their distinctive behavior in the spectrum of catanionic mixtures.

A Stereochemically Driven Supramolecular Polymerisation.

Anthracyclines self-assemble in water into dimers. In the presence of sufficiently high salt (NaCl) concentrations, solutions of the antibiotic doxorubicin, but not those of the closely related molecules daunomycin and epirubicin, turn into gels barely compatible with the presence of small oligomers. The use of spectroscopic, scattering, imaging and computational techniques, allowed light to be shed on the self-assembly process that triggered doxorubicin gelification. A complex picture emerged, with doxorubicin molecules assembled into long, highly chiral, supramolecular aggregates made of hundreds of units, showing redshifted fluorescence spectra, very short fluorescence lifetimes and small-angle X-ray scattering profiles compatible with long cylinders. The involvement of specific chemical groups and the need for a specific stereochemistry of the monomers in the formation of a hydrogen-bond network to stabilise the supramolecular aggregates was supported by molecular dynamics calculations. A salt-induced, temperature-dependent, cooperative nucleation-elongation supramolecular polymerisation of the doxorubicin molecules is deduced.

Twisted nanoribbons from a RGD-bearing cholic acid derivative.

In light of the biomedical interest for self-assembling amphiphiles bearing the tripeptide Arg-Gly-Gly (RGD), a cholic acid derivative was synthesized by introducing an aromatic moiety on the steroidal skeleton and the RGD sequence on the carboxylic function of its chain 17-24, thus forming a peptide amphiphile with the unconventional rigid amphiphilic structure of bile salts. In aqueous solution, the compound self-assembled into long twisted ribbons characterized by a very low degree of polydispersity in terms of width (≈25nm), thickness (≈4.5nm) and pitch (≈145nm). It was proposed that in the ribbon the molecules are arranged in a bilayer structure with the aromatic moieties in the interior, strongly involved in the intermolecular interaction, whereas the RGD residues are located at the bilayer-water interface. The nanostructure is significantly different from those generally provided by RGD-containing amphiphiles with the conventional peptide-tail structure, for which fibers with a circular cross-section were observed, and successfully tested as scaffolds for tissue regeneration. From previous work on the use of this kind of nanostructures, it is known that features like morphology, rigidity, epitope spacing and periodicity are important factors that dramatically affect cell adhesion and signaling. Within this context, the reported results demonstrate that bile salt-based peptide surfactants are promising building blocks in the preparation of non-trivial RGD-decorated nanoaggregates with well-defined morphologies and epitope distributions.

Unravelling the shape and structural assembly of the photosynthetic GAPDH-CP12-PRK complex from Arabidopsis thaliana by small-angle X-ray scattering analysis.

Oxygenic photosynthetic organisms produce sugars through the Calvin-Benson cycle, a metabolism that is tightly linked to the light reactions of photosynthesis and is regulated by different mechanisms, including the formation of protein complexes. Two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), form a supramolecular complex with the regulatory protein CP12 with the formula (GAPDH-CP122-PRK)2, in which both enzyme activities are transiently inhibited during the night. Small-angle X-ray scattering analysis performed on both the GAPDH-CP12-PRK complex and its components, GAPDH-CP12 and PRK, from Arabidopsis thaliana showed that (i) PRK has an elongated, bent and screwed shape, (ii) the oxidized N-terminal region of CP12 that is not embedded in the GAPDH-CP12 complex prefers a compact conformation and (iii) the interaction of PRK with the N-terminal region of CP12 favours the approach of two GAPDH tetramers. The interaction between the GAPDH tetramers may contribute to the overall stabilization of the GAPDH-CP12-PRK complex, the structure of which is presented here for the first time.

On the self-assembly of a tryptophan labeled deoxycholic acid.

Self-assembly of peptides and bile acids has been widely investigated because of their biological role and their potential as a tool for the preparation of nanostructured biomaterials. We herein report both the synthesis and the self-association behavior of a compound that combines the aggregation properties of bile acid- and amino acid-based molecules. The derivative has been prepared by introducing a L-tryptophan residue into the C-3 position of the deoxycholic acid skeleton and resulted in an amphoteric fluorescent labeled bile acid that shows a pH-dependent self-assembly. Under alkaline conditions it assembles into 28 nm diameter tubules, thus showing a completely different behavior compared to the precursor bile acid, which forms micelles under similar conditions. Upon heating the tubules break and turn into micelles, leading to an increase in the exposure to water of the tryptophan residue. On the other hand, in acidic solutions it aggregates into elongated micelles that further self-assemble forming a gel network, when an electrolyte is added.

Characterization of carbon nanotube dispersions in solutions of bile salts and derivatives containing aromatic substituents.

Bile salts (BS) are known to solubilize high weight fractions of carbon nanotubes (CNTs) in aqueous solutions. Here, the efficiency of derivatives of bile salts (BSDs) containing aromatic substituents in dispersing single-wall CNTs (SWCNTs) has been investigated in order to check whether the presence of aromatic residues, because of their affinity toward carbon nanotube surfaces, determines improvements of the BS dispersion efficiency (DE). Electric arc and CoMoCAT SWCNTs were analyzed. The results, reported for the two surfactant concentrations of 0.06 and 1.0 wt %, show that the DE of BSDs depends on the position, orientation, and structure of the introduced aromatic residues. In the case of the CoMoCAT SWCNTs, at low surfactant concentration a DE improvement is observed in BSDs where the aromatic residue is linked either to carbon 3, located on the rigid four-ring system, or to the side chain. For the latter, this improvement is also enhanced in double-charge derivatives and kept at high surfactant concentration. It was also observed that at low concentrations of surfactant, the DE values of BSs and BSDs are usually larger than those of the more conventional detergent sodium dodecylsulfate.

A single amino-acid substitution allows endo-polygalacturonase of Fusarium verticillioides to acquire recognition by PGIP2 from Phaseolus vulgaris.

Polygalacturonases (PGs) are secreted by phytopathogenic fungi to degrade the plant cell wall homogalacturonan during plant infection. To counteract Pgs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs) that slow down fungal infection and defend cell wall integrity. PGIPs favour the accumulation of oligogalacturonides, which are homogalacturonan fragments that act as endogenous elicitors of plant defence responses. We have previously shown that PGIP2 from Phaseolus vulgaris (PvPGIP2) forms a complex with PG from Fusarium phyllophilum (FpPG), hindering the enzyme active site cleft from substrate. Here we analyse by small angle X-ray scattering (SAXS) the interaction between PvPGIP2 and a PG from Colletotrichum lupini (CluPG1). We show a different shape of the PG-PGIP complex, which allows substrate entry and provides a structural explanation for the different inhibition kinetics exhibited by PvPGIP2 towards the two isoenzymes. The analysis of SAXS structures allowed us to investigate the basis of the inability of PG from Fusarium verticilloides (FvPG) to be inhibited by PvPGIP2 or by any other known PGIP. FvPG is 92.5% identical to FpPG, and we show here, by both loss- and gain-of-function mutations, that a single amino acid site acts as a switch for FvPG recognition by PvPGIP2.

Drug-loaded nanoparticles and supramolecular nanotubes formed from a volatile microemulsion with bile salt derivatives.

The main objective of this study was to form nanoparticles of a model hydrophobic drug, celecoxib, from a volatile microemulsion stabilized by a bile salt derivative. Nanoparticles were obtained by conversion of the microemulsion nanodroplets with the dissolved drug into solid nanometric particles. The use of bile salt derivatives as the surfactants for the formation of a microemulsion enabled significantly higher loading of the drug in both the microemulsion and nanoparticles, compared with the native bile salt. In addition, superior stability of the particles was achieved with the bile salt derivatives, and drug crystallization was inhibited. Interestingly, differences in particle stability and crystallization inhibition were observed between two bile salt derivatives differing only by one hydroxyl group on the bile salt backbone, indicating the delicate balance of interactions in the system. For one of the derivatives, upon dispersion of the nanoparticles in water, they spontaneously arranged into well-defined elongated nanometric tubules as detected and attested by cryo-TEM. It was found that the drug present in nanoparticles induces formation of the nanotubes.

Crystal structure of head-to-head dimers of cholic and deoxycholic acid derivatives with different symmetric bridges.

The crystal structure of three head-to-head dimers (having two cholic acid or deoxycholic acid units) linked at carbon atoms C3 by aromatic or alkyl bridges is studied. An internal coordinates system is necessary for describing the relative orientation in the space of the two bile acid residues. Five angles (three torsion and two common ones) are necessary for defining the relative position of both steroid residues in space. Carbon atoms C3 (which always carries a α-hydroxy group in natural bile acids), and C10 and C13 (which always carry ß-methyl groups) of each steroid residue are suitable for this purpose. Furthermore, the distance between each C3 carbon atoms of both steroid residues will allow one to locate the steroids in space. The three dimers selected provide a large range of values for these angles. The packing, hydrogen bond network, and location of guest in the three crystals are discussed.

Formation of tubules by p-tert-butylphenylamide derivatives of chenodeoxycholic and ursodeoxycholic acids in aqueous solution.

The formation of tubules by p-tert-butylphenylamide derivatives of chenodeoxycholic and ursodeoxycholic acids in aqueous solution is investigated. The critical aggregation concentrations of the new surfactants are much lower than those of ursodeoxycholate and chenodeoxycholate, indicating the enhanced surfactant properties resulting by the presence of the hydrophobic p-tert-butylphenyl group. The molecular areas at the air-water interface suggest the formation of monolayer films with molecules upright oriented. The shape of the aggregates was investigated by TEM. The main structure present in solution corresponds to tubules. The estimated value for the wall thickness of tubules suggests that a bilayer structure is formed. Host of positively charged latex beads by tubules suggests that their inner and outer surfaces are negatively charged. The acid form of the chenodeoxycholate derivative was recrystallized from toluene and its crystal structure analyzed.

Structural resolution of the complex between a fungal polygalacturonase and a plant polygalacturonase-inhibiting protein by small-angle X-ray scattering.

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.

Supramolecular structures generated by a p-tert-butylphenylamide derivative of deoxycholic acid. From planar sheets to tubular structures through helical ribbons.

The formation of supramolecular structures initiated by a p-tert-butylphenylamide derivative of deoxycholic acid (Na-t-butPhDC) is investigated. At 1.18 mM concentration of Na-t-butPhDC and 37 degrees C, initial flat ribbons are observed which self-transform into helical ribbons (with a mean pitch angle of 47 +/- 6 degrees) which finally originate molecular tubes with an external diameter of 241 +/- 28 nm. Most of the molecular tubes show helical markings with a pitch angle value of 45 +/- 4 degrees, in full agreement with predictions of simple models based on chiral elastic properties of the membrane. A lateral association mechanism is proposed to account for the growth of the external diameter (from 225 +/- 32 to 546 +/- 59 nm) of tubes with time at 3.99 mM.

Sodium glycodeoxycholate and glycocholate mixed aggregates in gas and solution phases.

This paper deals with electrospray ionization mass spectrometry (ESIMS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS) measurements in order to provide information on the existence, aggregation, composition, and structure of the two-component aggregates of sodium glycocholate (NaGC) and sodium glycodeoxycholate (NaGDC) in the gas and solution phases. Five samples, containing 100% NaGC and 100% NaGDC, and NaGDC/NaGC molar ratios of 3 (75D), 1 (50D), and 1/3 (25D), have been analyzed by ESIMS in positive-ion detection mode starting from 10(-3) and 10(-2) M total bile salt concentration in aqueous solutions. Generally, dimers or trimers prevail in the 100% NaGC or NaGDC samples, respectively, as observed in the preceding one-component ESIMS measurements and in agreement with the proposed micellar aggregate structures in aqueous solution. Moreover, it is observed that the composition of multimers in the samples 75D, 50D, and 25D deviates from the one expected on the basis of a random association of the monomers, the NaGDC contribution generally prevailing on the NaGC one. It happens also under the same percentage condition (50D sample), in agreement with a greater aggregation ability of NaGDC with respect to NaGC. SAXS and DLS data were recorded on six samples containing a NaGC+NaGDC 40 mM total concentration, one bile salt having 40, 32, 24, 16, 8, and 0 mM concentration and the other the complementary one, keeping constant the NaCl concentration (0.6 M). The NaGDC 40 mM sample presents SAXS curves in agreement with a cylindrical shape of the aggregates as shown in a previous paper. For the bile salt mixtures, the progressive decrease of the sizes and change of the aggregate morphology, toward a globular-like geometry, are observed by increasing the NaGC fraction, thus confirming the hypothesis about the ability of trihydroxy salts to inhibit the growth of dihydroxy salt aggregates. Fits on the basis of cylindrical model can be accomplished for all the SAXS spectra, however, when the extracted cylinder parameters are used to estimate theoretical hydrodynamic radii a reasonable agreement is obtained only for the samples at high fraction of NaGDC (NaGDC>or=24 mM).

About the albumin structure in solution: cigar Expanded form versus heart Normal shape.

A structural comparison between the Normal and the Expanded isomers of the human serum albumin has been carried out by using small angle X-ray scattering (SAXS) and light scattering (LS) techniques. Geometrical bodies, recovered structures (GA_STRUCT code) and rigid body modeling (CRYSOL and BUNCH software) were used to obtain low-resolution 3D structures from one-dimensional scattering patterns. These restored shapes were also exploited to perform a correlation between SAXS and LS data. By attempting a detailed description of globular and unfolded protein structures in solution, we tried to propose a suitable approach to follow the path of folding/unfolding processes and to isolate and characterize possible partially folded intermediate states.

Human serum albumin unfolding: a small-angle X-ray scattering and light scattering study.

We report a study on the unfolding behavior of the most abundant protein contained in plasma, the fatted and defatted human serum albumin, in denaturing conditions induced by guanidine hydrochloride. Low-resolution three-dimensional structures are reconstructed from the one-dimensional (1D) small-angle X-ray scattering patterns, and they are correlated with the parameters obtained from static and dynamic light scattering experiments. The unfolding process is pointed out by both ab initio and rigid body fitting methods which highlight a stepwise evolution of the protein structure toward open conformations. The superpositions of the 3D structures provided independently by the two methods show very good agreements. The hydrodynamic radii estimated for the protein best fitting conformations are in satisfactory agreement with the experimental ones. The results show that the unfolding process is consistent with previous spectroscopic studies which suggest a multistep unfolding pathway. In particular, a scheme in which domains III and II are opened in sequence and the presence of two intermediates are evidenced is presented.

Early stages of formation of branched host-guest supramolecular polymers.

A structural characterization of host-guest supramolecular copolymers, formed by an adamantane dimer and two beta-cyclodextrin trimers in aqueous solution, has been carried out by combining small angle X-ray scattering and light scattering experiments. A shape-reconstruction method was applied to the SAXS data to obtain relatively high-resolution conformation information, and a correlation with the experimental dynamic light scattering results was performed, by estimating the hydrodynamic radii of the reconstructed shape through a shell model method. When applied on the solutions of the trimers, the analysis provides a globular reconstructed shape with a hydrodynamic radius in agreement with the experimental one. For the polymers, elongated structures were inferred which grow both in length and in cross section by increasing the concentration. Depending on the beta-cyclodextrin trimer employed in the polymer preparation, polymerization degrees ranging between roughly 7 and 14 or 9 and 22 were obtained in the concentration range 4.00-10.0 or 3.10-6.60 mM of the trimer (6.00-15.0 or 4.65-9.90 mM of the dimer). Aggregation schemes were proposed accounting for the formation of hyperbranched, linear, and network like polymers. The experimental results are not far from those expected on the basis of the aggregation in hyperbranched structure, for which the growth of elongated aggregates can be predicted in the early stages of the polymerization. However, the coexistence of the other structures, in particular of the linear one, cannot be ruled out.