Claudia Leggio

Catanionic Tubules with Tunable Charge

Thethree-dimensionalstructureswithnanoscopicdimensionsthat are yielded by the self-assembly of lipids and surfactantsare of particular interest for their applications in nano-technology.Intheseapplications,thepossibilityofcontrollingthe charge of the particles allows the regulation of funda-mental aspects, such as the ability of the particles to loadmolecules (drugs, DNA, proteins, etc.), to aggregate, and topenetrate membranes. Within the possible surfactant supra-molecular architectures, tubular structures have recentlydrawn much research interest.

Kinetics of formation of supramolecular tubules of a sodium cholate derivative

We report a kinetic study of the supramolecular tubule formation of the bile salt derivative [3β,5β,7α,12α]-3-(4-t-butylbenzoilamine)-7,12-dihydroxycholan-24-oic acid sodium salt (Na-tbutPhC). At high bicarbonate buffer concentration (pH∼10) this salt shows gelator properties. Starting from gels or viscous solutions, the tubule formation is triggered by increasing the temperature beyond the critical value of 34–36 °C. For gels, when the process takes place, the transition to sols occurs. The process is easily triggered and can be followed by several techniques. We used static light scattering (SLS), circular dichroism (CD), small angle X-ray scattering (SAXS) along with transmission electron (TEM) and optical microscopies. The CD results show that fibrils with a clockwise arrangement of the bile salt derivative are present in the samples at room temperature. When the tubule formation starts, evolutions of the CD and SLS profiles are observed indicating that the formation process begins with the aggregation of the fibrils accompanied by a simultaneous peculiar reciprocal reorientation of the surfactant molecules. After that, as pointed out by the long time evolution of the curves, a slow transformation towards the final well defined tubules occurs, involving an adjustment of the molecular packing. In the meanwhile, the slow ordering of the tubule walls in well spaced layers takes place, as inferred by SAXS. The TEM images show that short disordered tubules are formed, because of the aggregation of fibrils, in the beginning. Moreover they highlight a final elongation of the tubules taking place without a further aggregation of fibrils. Optical microscopy frames, collected during the process, point out that the tubules grow singly even at quite a high concentration, thus supporting the data interpretation.

Urea-Induced Denaturation Process on Defatted Human Serum Albumin and in the Presence of Palmitic Acid

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.

Human serum albumin binding ibuprofen: A 3D description of the unfolding pathway in urea

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.

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.

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.

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.

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).

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.