RESUMEN
Amphipathic peptides are attractive building blocks for the preparation of self-assembling, bio-inspired, and stimuli responsive nanomaterials with pharmaceutical interest. The bioavailability of these materials can be improved with the insertion of d amino acid residues to avoid fast proteolysis in vivo. With this knowledge, a new lauroyl peptide consisting of a sequence of glycine, glycine, d-serine, and d-lysine was designed. In spite of its simple sequence, this lipopeptide self-assembles into spherical micelles at acid pH, when the peptide moiety adopts disordered conformations. Self-aggregates reshape toward fibers at basic pH, following the conformational transition of the peptide region from random coil to ß-sheet. Finally, hydrogels are achieved at basic pH and higher concentrations. The transition from random coil to ß-sheet conformation of the peptide headgroup obtained by increasing pH was monitored by circular dichroism and vibrational spectroscopy. A structural analysis, performed by combining dynamic light scattering, small-angle X-ray scattering, transmission electron microscopy, and molecular dynamic simulations, demonstrated that the transition allows the self-assemblies to remodel from spherical micelles to rodlike shapes, to long fibers with rectangular cross-section and a head-tail-tail-head structure. The viscoelastic behavior of the hydrogels formed at the highest pH was investigated by rheology measurements.
Asunto(s)
Hidrogeles , Péptidos , Aminoácidos , Dicroismo Circular , Concentración de Iones de HidrógenoRESUMEN
To study the formation and characterize the structure of mixed complexes of oppositely charged block copolymers and surfactants are of great significance for practical applications, e.g., in drug carrier formulations that are based on electrostatically assisted assembly. In this context, biocompatible block copolymers and biosurfactants (like bile salts) are particularly interesting. In this work, we report on the co-assembly in dilute aqueous solution between a cationic poly(N-isopropyl acryl amide) (PNIPAM) diblock copolymer and the oppositely charged bile salt surfactant sodium deoxycholate at ambient temperature. The cryogenic transmission electron microscopy (cryo-TEM) experiments revealed the co-existence of two types of co-assembled complexes of radically different morphology and inner structure. They are formed mainly as a result of the electrostatic attraction between the positively charged copolymer blocks and bile salt anions and highlight the potential of using linear amphiphilic block copolymers as bile salt sequestrants in the treatment of bile acid malabsorption and hypercholesterolemia. The first complex of globular morphology has a coacervate core of deoxycholate anions and charged copolymer blocks surrounded by a PNIPAM corona. The second complex has an intriguing tape-like supramolecular morphology of several micrometer in length that is striped in the direction of the long axis. A model is presented in which the stretched cationic blocks of several block copolymers interact electrostatically with the bile salt molecules that are associated to form a zipper-like structure. The tape is covered on both sides by the PNIPAM chains that stabilize the overall complex in solution. In addition to cryo-TEM, the mixed system was investigated in a range of molar charge fractions at a constant copolymer concentration by static light scattering, small angle X-ray scattering, and electrophoretic mobility measurements.
RESUMEN
An approach for tailoring self-assembled tubular structures is described. By controlling the relative composition of a two-component surfactant mixture comprising the natural bile salt lithocholate and its bolamphiphilic derivative, it was possible to finely tune the nanotube cross-section of the mixed tubular aggregates that self-associated spontaneously in aqueous solution at pHâ 12. The diameter was found to vary up to 50% when the stoichiometric ratio of the two bile salts was changed. The tuning of supramolecular nanochannels with such remarkable precision is of significant interest for technological applications of these materials.
Asunto(s)
Ácidos y Sales Biliares/química , Nanotubos/química , Tensoactivos/química , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
The introduction of a mannose residue on carbon 3 of lithocholic acid gives rise to an asymmetric and rigid bolaamphiphilic molecule, which self-assembles in water to form elongated tubular aggregates with an outer diameter of about 20 nm. These tubular structures display a temporal evolution, where the average tube diameter decreases with time, which can be followed by time-resolved small-angle X-ray scattering experiments. Cryogenic transmission electron microscopy images collected as a function of time show that at short times after preparation tubular scrolls are formed via the rolling of layers, after which a complex transformation of the scrolls into single-walled tubules takes place. At long time scales, a further evolution occurs where the tubules both elongate and become narrower. The observed self-assembly confirms the tendency of bile acids and their derivatives to form supramolecular aggregates with an ordered packing of the constituent molecules. It also demonstrates that scrolls can be formed as intermediate structures in the self-assembly process of monodisperse single-walled tubules.
RESUMEN
Nanoparticles with an internal structure have been prepared by dispersing under dilute conditions poly(acrylic acid) with a polymerization degree n = 6000 (PAA6000) together with a cationic surfactant hexadecyltrimethylammonium hydroxide (C16TAOH) and the non-ionic surfactant penta(ethylene glycol) monododecyl ether (C12E5) in water. The nanoparticles are formed at different mixing ratios in the corresponding two-phase regions (liquid crystalline phase/dilute isotropic phase) of the C16TAPA6000 complex salt/C12E5/water ternary phase diagram. The particles consist of polyacrylate PA6000 polyions, C16TA+ surfactant ions, and C12E5. Their internal ordering was identified by small-angle X-ray scattering (SAXS) to be either bicontinuous cubic with the Ia3d crystallographic space group or normal hexagonal depending upon the amount of C12E5. The bicontinuous cubic phase, to our knowledge never observed before in polyelectrolytesurfactant particle systems, was inferred by SAXS experiments. The data also showed that this structure is thermoresponsive in a reversible manner. The bicontinuous cubic space group transforms from Ia3d to Im3m as the temperature decreases from 25 to 15 °C. According to dynamic light scattering and electrophoretic mobility measurements, the particles have a well-defined size (apparent hydrodynamic radii RH in the range of 88140 nm) and carry a positive net charge. The size of the nanoparticles is stable up to 1 month. The faceted nanoparticles are visualized by cryogenic transmission electron microscopy that also reveals their coexistence with thread-like C12E5 micelles.
RESUMEN
We report a study on the unfolding behavior of the most abundant protein contained in plasma, human serum albumin. The unfolding mechanisms in denaturing conditions induced by urea are studied for the defatted form (HSA) and for the palmitic acid:albumin (HSAPalm) complex. We employed the singular value decomposition method to determine the minimum number of structural states present in the unfolding processes. Low-resolution three-dimensional structures are reconstructed from the one-dimensional small-angle X-ray scattering patterns and 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 that highlight a stepwise evolution of the protein structure toward open conformations. The superimpositions 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 HSA unfolding process is consistent with previous spectroscopic studies that suggest a multistep unfolding pathway. In particular, a scheme in which domains I and II are opened in sequence and the presence of two intermediates are evidenced is presented. The opening sequence is different from that found using guanidine hydrochloride as denaturant agent. The stabilizing role of the fatty acids in the urea denaturation process is evident. The palmitic acid ligand strongly stabilizes the protein, which remains in the native form up to high denaturant concentrations. In this case, the unfolding process is characterized by a single-step mechanism.
Asunto(s)
Ácido Palmítico/química , Albúmina Sérica/química , Urea/farmacología , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Humanos , Luz , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica/efectos de los fármacos , Reproducibilidad de los Resultados , Dispersión de Radiación , Dispersión del Ángulo Pequeño , Espectrometría de Fluorescencia , Difracción de Rayos XRESUMEN
The protein Human Serum Albumin (HSA) is known to undergo conformational transitions towards partially unfolded forms triggered by acidification below pH 4.5. The extent of Fatty Acids (FA) binding has been thought to have an impact on the conformational equilibrium between the native and acid forms and to be a possible explanation for the observation of more than one band in early electrophoretic migration experiments at pH 4. We compared the acid-induced unfolding processes of commercial FA-free HSA, commercial "fatted" HSA and FA-HSA complexes, prepared at FA:HSA molar ratios between 1 and 6 by simple mixing and equilibration. We used a method for continuous acidification based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, and followed the average protein changes by the blue shift of the intrinsic fluorescence emission and by performing a small angle X-ray scattering analysis on selected samples. The method also allowed for continuous monitoring of the increase of turbidity and laser light scattering of the protein samples related to the release of the insoluble ligands with acidification. Our results showed that the presence of FA interacting with albumin, an aspect often neglected in biophysical studies, affects the conformational response of the protein to acidification, and slightly shifts the loss of the native shape from pH 4.2 to pH 3.6. This effect increased with the FA:HSA molar ratio so that with three molar equivalents a saturation was reached, in agreement with the number of high-affinity binding sites reported for the FA. These findings confirm that a non-uniform level of ligand binding in an albumin sample can be an explanation for the early-observed conformational heterogeneity at pH 4.
Asunto(s)
Ácidos/química , Ácidos Grasos/química , Conformación Proteica , Albúmina Sérica Humana/química , Ácidos Grasos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Isomerismo , Unión Proteica , Desplegamiento Proteico , Dispersión del Ángulo Pequeño , Albúmina Sérica Humana/metabolismo , Espectrometría de Fluorescencia , Difracción de Rayos XRESUMEN
The most abundant plasma protein, human serum albumin (HSA), is known to undergo several conformational transitions in an acidic environment. To avoid buffer effects and correlate global and local structural changes, we developed a continuous acidification method and simultaneously monitored the protein changes by both small-angle scattering (SAXS) and fluorescence. The progressive acidification, based on the hydrolysis of glucono-δ-lactone from pH 7 to pH 2.5, highlighted a multistep unfolding involving the putative F form (pH 4) and an extended and flexible conformation (pH < 3.5). The scattering profile of the F form was extracted by component analysis and further 3D modeled. The effect of acid unfolding at this intermediate stage was assigned to the rearrangement of the three albumin domains drifting apart toward a more elongated conformation, with a partial unfolding of one of the outer domains. To test the stabilizing effect of fatty acids, here palmitic acid, we compared the acid unfolding process of albumin with and without ligand. We found that when binding the ligand, the native conformation was favored up to lower pH values. Our approach solved the problem of realizing a continuous, homogeneous, and tunable acidification with simultaneous characterization applicable to study processes triggered by a pH decrease.
Asunto(s)
Ácido Palmítico/química , Albúmina Sérica Humana/química , Sitios de Unión , Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Estabilidad Proteica , Desplegamiento Proteico , Dispersión del Ángulo Pequeño , Factores de Tiempo , Difracción de Rayos XRESUMEN
The most abundant plasma protein, human serum albumin (HSA), plays a key part in the body's antioxidant defense against reactive species. This study was aimed at correlating oxidant-induced chemical and structural effects on HSA. Despite the chemical modification induced by the oxidant hypochlorite, the native shape is preserved up to oxidant/HSA molar ratio <80, above which a structural transition occurs in the critical range 80-120. This conformational variation involves the drifting of one of the end-domains from the rest of the protein and corresponds to the loss of one-third of the α-helix and a net increase of the protein negative charge. The transition is highly reproducible suggesting that it represents a well-defined structural response typical of this multidomain protein. The ability to tolerate high levels of chemical modification in a folded or only partially unfolded state, as well as the stability to aggregation, provides albumin with optimal features as a biological buffer for the local formation of oxidants.
Asunto(s)
Ácido Hipocloroso/síntesis química , Albúmina Sérica/química , Humanos , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Oxidación-Reducción , Conformación Proteica , Albúmina Sérica/metabolismoRESUMEN
The crystal structure of a Li(+) salt of a glucosyl derivative of lithocholic acid (lithium 3α-(α-d-glucopyranosyl)-5ß-cholan-24-oate) has been solved. The crystal belongs to the orthorhombic system, P212121 spatial group, and includes acetone and water in the structure with a 1:1:2 stoichiometry. Monolayers, having a hydrophobic interior and hydrophilic edges, are recognized in the crystal structure. Li(+) is coordinated to three hydroxyl groups of three different glucose residues, with two of them belonging to the same monolayer. A fourth molecule, located in this monolayer, is involved in the coordination of the cation through the carboxylate ion by an electrostatic interaction, thus completing a distorted tetrahedron. All Li(+)-oxygen distances values are very close to the sum of the ionic radius of Li(+) and van der Waals radius of oxygen. Each steroid molecule is linked to other five steroid molecules through hydrogen bonds. Water and acetone are also involved in the hydrogen bond network. A hierarchical organization can be recognized in the crystal, the helical assembly along 21 screw axes being left-handed.
Asunto(s)
Cristalografía por Rayos X/métodos , Litio/química , Ácido Litocólico/química , Modelos MolecularesRESUMEN
The unfolding pathway of the defatted human serum albumin (HSA) binding ibuprofen and propofol has been studied by using small-angle X-ray scattering (SAXS) and the support of circular dichroism data. A set of HSA solutions with urea concentrations between 0.00 and 9.00 M was analyzed, and the singular value decomposition method applied to the complete SAXS data set allowed us to distinguish four different states in solution. Besides the native and unfolded forms, two intermediates I1 and I2 have been identified, and the low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. The I1 structure was characterized by only one open domain (domain I, which does not host a binding site for either of the ligands), whereas I2 presents only one closed domain (domain III). A direct comparison with the unfolding pathway of the HSA:Ibu complex (Galantini et al. Biophys. Chem. 2010, 147, 111-122) pointed out that the presence of propofol as a second ligand, located in subdomain IIIB, leads to the appearance of an intermediate with two closed domains (domains II and III), which are those that accommodate the ligands. Moreover, the equilibrium between I2 and the unfolded form is slightly shifted toward higher urea concentrations. These results suggest that the cobinding significantly hinders the unfolding process.
Asunto(s)
Ibuprofeno/farmacología , Propofol/farmacología , Pliegue de Proteína/efectos de los fármacos , Albúmina Sérica/química , Urea/química , Anestésicos Intravenosos/química , Anestésicos Intravenosos/farmacología , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Sitios de Unión , Dicroismo Circular , Humanos , Ibuprofeno/química , Propofol/química , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Dispersión del Ángulo Pequeño , Albúmina Sérica/metabolismo , Espectrometría de Fluorescencia , Difracción de Rayos XRESUMEN
Biocompatible molecules that undergo self-assembly are of high importance in biological and medical applications of nanoscience. Peptides and bile acids are among the most investigated due to their ability to self-organize into many different, often stimuli-sensitive, supramolecular structures. With the aim of preparing molecules mixing the aggregation properties of bile acid and amino acid-based molecules, we report on the synthesis and self-association behavior of two diastereomers obtained by substituting a hydroxyl group of cholic acid with a l-phenylalanine residue. The obtained molecules are amphoteric, and we demonstrate that they show a pH-dependent self-assembly. Both molecules aggregate in globular micelles at high pH, whereas they form tubular superstructures under acid conditions. Unusual narrow nanotubes with outer and inner cross-section diameters of about 6 and 3 nm are formed by the derivatives. The diasteroisomer with α orientation of the substituent forms in addition a wider tubule (17 nm cross-section diameter). The ability to pack in supramolecular tubules is explained in terms of a wedge-shaped bola-form structure of the derivatives. Parallel or antiparallel face-to-face dimers are hypothesized as fundamental building blocks for the formation of the narrow and wide nanotubes, respectively.
Asunto(s)
Ácidos y Sales Biliares/química , Ácidos Cólicos/química , Péptidos/química , Fenilalanina/química , Dicroismo Circular , Concentración de Iones de Hidrógeno , Micelas , Microscopía de Fuerza Atómica , Péptidos/metabolismo , Tensión Superficial , TemperaturaRESUMEN
An amino acid-substituted bile acid forms tubular aggregates with inner and outer diameters of about 3 and 6 nm. The diameters are unusually small for surfactant self-assembled tubes. The results enhance the spectrum of applications of supramolecular tubules and open up possibilities for investigating a novel class of biological amphiphiles.
Asunto(s)
Aminoácidos/química , Ácidos y Sales Biliares/química , Nanotubos/química , Fenilalanina/química , Tensoactivos/química , Modelos Moleculares , Conformación MolecularRESUMEN
Small angle X-ray scattering (SAXS) technique, supported by light scattering measurements and spectroscopic data (circular dichroism and fluorescence) allowed us to restore the 3D structure at low resolution of defatted human serum albumin (HSA) in interaction with ibuprofen. The data were carried out on a set of HSA solutions with urea concentrations between 0.00 and 9.00M. The Singular Value Decomposition method, applied to the complete SAXS data set allowed us to distinguish three different states in solution. In particular a native conformation N (at 0.00M urea), an intermediate I1 (at 6.05M urea) and an unfolded structure U (at 9.00M urea) were recognized. The low-resolution structures of these states were obtained by exploiting both ab initio and rigid body fitting methods. In particular, for the protein without denaturant, a conformation recently described (Leggio et al., PCCP, 2008, 10, 6741-6750), very similar to the crystallographic heart shape, with only a slight reciprocal movement of the three domains, was confirmed. The I1 structure was instead characterized by only a closed domain (domain III) and finally, the recovered structure of the U state revealed the characteristic feature of a completely open state. A direct comparison with the free HSA pointed out that the presence of the ibuprofen provokes a shift of the equilibrium towards higher urea concentrations without changing the unfolding sequence. The work represents a type of analysis which could be exploited in future investigations on proteins in solution, in the binding of drugs or endogenous compounds and in the pharmacokinetic properties as well as in the study of allosteric effects, cooperation or anticooperation mechanisms.
Asunto(s)
Ibuprofeno/química , Albúmina Sérica/química , Urea/química , Sitios de Unión , Humanos , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Dispersión del Ángulo Pequeño , Urea/farmacología , Difracción de Rayos XRESUMEN
A quite unexpected sevenfold coordination of the hydrated Hg(II) complex in aqueous solution is revealed by an extensive study combining X-ray absorption spectroscopy (XAS) and quantum mechanics/molecular dynamics (QM/MD) calculations. As a matter of fact, the generally accepted octahedral solvation of Hg(II) ion cannot be reconciled with XAS results. Next, refined QM computations point out the remarkable stability of a heptacoordinated structure with C2 symmetry, and long-time MD simulations by new interaction potentials including many-body effects reveal that the hydrated complex has a quite flexible structure, corresponding for most of the time to heptacoordinated species. This picture is fully consistent with X-ray absorption near-edge structure experimental data which unambiguously show the preference for a sevenfold instead of a sixfold coordination.