1,720,975 research outputs found
About the albumin structure in solution: cigar Expanded form versus heart Normal shape
No full textA 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
No full textWe 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
Correlation between small-angle X-ray scattering spectra and apparent diffusion coefficients in the study of structure and interaction of sodium taurodeoxycholate micelles
No full textSmall-Angle X-ray Scattering (SAXS) and Dynamic Light Scattering (DLS) measurements were carried out on aqueous micellar solutions of the ionic biological detergent sodium taurodeoxycholate (NaTDC). Apparent diffusion coefficients (D app) and SAXS spectra of NaTDC 0.1 M solutions at different ionic strengths (0.10.3 M NaCl) were reported. A comparative analysis of SAXS spectra and D app data was performed to infer information on particle structure and interaction potential. Uniform particles with a spherical, an oblate, and a prolate symmetry were used to model the micelles in the data interpretation. A hard-core interaction shell of suitable thickness and a screened Coulomb potential of the electric double layer (EDL potential) were alternatively used to represent the long-range repulsive tail of the interaction potential. The Percus Yevick and the Rescaled Mean Spherical Approximation were applied. To compare the data of the two techniques, for each sample, a D app was calculated from the SAXS best-fitting geometrical parameters and interparticle structure factor of the micelles. Hence, a fitting procedure involving both the scattering and D app data was performed. The interpretation of SAXS spectra does not allow the discrimination between the oblate and the prolate symmetries of the aggregates. On the other hand, the comparison of calculated and experimental D app values indicates that the prolate ellipsoid is better suited to represent the micelle shape. Moreover, the agreement between calculated and experimental D app values is sensitively better at the lowest NaCl concentration when the EDL potential is used. A rodlike micellar growth and a progressive screening of the electrostatic interactions is testified by the trends of best-fitting parameters as a function of the added electrolyte. © 2005 American Chemical Society
Small-angle X-ray scattering and light scattering on lysozyme and sodium glycocholate micelles
No full textSmall-angle X-ray scattering (SAXS) together with static (SLS) and dynamic light scattering (DLS) measurements were carried out on aqueous solutions of lysozyme (LY) and of the ionic biological detergent sodium glycocholate (NaGC). Apparent diffusion coefficients (D-app), excess Rayleigh ratio, and SAXS spectra were measured for 0.1 M NaGC solutions at different ionic strengths (0.05-0.30 M NaCl). The same data were collected for LY in sodium acetate buffer 50 mM without and with 92 mM NaCl as a function of protein concentration (10-80 g L-1). A correlated analysis of SLS data and SAXS spectra was first tested on the LY samples and then extended to the interpretation of the NaGC data to infer information on particle structure and interaction potential. A hard-core (HC) interaction shell of uniform thickness, a screened Coulomb potential of the electric double layer (EDL) or the complete DLVO potential were alternatively used to represent the long-range tail of the interaction potential. Whenever an essentially repulsive tail is expected, all the representations give reasonable results, but the data analysis does not allow the discrimination between the oblate and the prolate symmetries of the NaGC aggregates. The DLVO model allows the interpretation of the data even when the attractive component determines the tail character. With this model an overall fit of the micelle data at all the NaCl concentrations was successfully performed by assuming a simple spherical symmetry of the micelles and invariant values of their ionization degree and Hamaker constant, thus considering just the screening effect of the added electrolyte. Whatever model is used, the results point out that the aggregates are quite hydrated (26-38 water molecules per monomer) and very slightly grow by increasing the NaCl concentration. When spherical symmetry is assumed the aggregate radii for all the samples fall in the range 15-16 angstrom. From the SAXS and SLS, best fitting geometrical parameters, and interparticle structure factor, a D-app value was calculated for each sample. An excellent consistence is achieved for LY results. On the contrary, calculated D-app values systematically lower than the experimental values are always obtained for the NaGC micelles. Micelle polydispersity and internal dynamics seem to be the most probable reasons of the bad agreement
Human serum albumin binding ibuprofen: A 3D description of the unfolding pathway in urea
No full textSmall 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 9.00 M. 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 0.00 M urea), an intermediate 11 (at 6.05 M urea) and an unfolded structure U (at 9.00 M 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 11 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. (C) 2010 Elsevier B.V. All rights reserved
Urea-Induced Denaturation Process on Defatted Human Serum Albumin and in the Presence of Palmitic Acid
No full textWe report a study on the unfolding behavior of the most abundant protein contained in plasma, human serumalbumin. The unfolding mechanisms in denaturing conditions induced by urea are studied for the defattedform (HSA) and for the palmitic acid:albumin (HSAPalm) complex. We employed the singular valuedecomposition 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-rayscattering patterns and are correlated with the parameters obtained from static and dynamic light scatteringexperiments. The unfolding process is pointed out by both ab initio and rigid body fitting methods that highlighta stepwise evolution of the protein structure toward open conformations. The superimpositions of the 3Dstructures provided independently by the two methods show very good agreements. The hydrodynamic radiiestimated 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 suggesta multistep unfolding pathway. In particular, a scheme in which domains I and II are opened in sequence andthe presence of two intermediates are evidenced is presented. The opening sequence is different from thatfound using guanidine hydrochloride as denaturant agent. The stabilizing role of the fatty acids in the ureadenaturation process is evident. The palmitic acid ligand strongly stabilizes the protein, which remains in thenative form up to high denaturant concentrations. In this case, the unfolding process is characterized by asingle-step mechanism
Structure of sodium glycodeoxycholate micellar aggregates from small-angle X-ray scattering and light-scattering techniques
No full textSmall-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) measurements were accomplished on sodium glycodeoxycholate (NaGDC) aqueous electrolyte solutions as a function of NaGDC and NaCl concentrations with the aim to determine with satisfactory approximation the NaGDC micellar aggregate structure at a gross molecular level, assuming monodispersity. Different conditions of interparticle interactions by varying ionic strength ( NaCl concentration from 0 to 0.70 M) and NaGDC concentration (from 0.02 to 0.10 M) were studied. Smeared scattering intensities and electron pair distance distribution functions, radii of gyration, and aggregate heights are in satisfactory agreement with the corresponding functions calculated using a 21 helix as model. It is formed by trimers, each one composed by three NaGDC and nine H2O molecules related by a 3-fold rotation axis, and can be described by a hollow cylinder, probably filled by water molecules, characterized by a conventional radius of 23.7 angstrom and a trimer repeat along the helical axis of 3.6 angstrom. The helix is considerably inhomogeneous since the volume of the cylinder occupied by NaGDC molecules is less than one-third of the total. On the other hand, calculations performed with the average radial electronic density of the helix without water molecules or totally filled by water molecules (a NaGDC/H2O molecular ratio of about 1/50) or by using a three-shell average radial electronic density, independently evaluated on absolute scale, do not show significant differences, thus supporting the helical model. The aggregate size increases for all the samples by increasing either the NaCl or NaGDC concentration. The NaGDC low concentration (0.02 M) samples with NaCl within the range 0.30-0.70 M are characterized by short cylindrical aggregates that do not give rise to sensible interference effects. This assertion is supported by the satisfactory fit between the observed apparent mean hydrodynamic radii and the calculated ones by means of the method of Ortega and Garcia de la Torre (J. Chem. Phys. 2003, 119, 9914), valid for rods with a length-to-diameter ratio >= 0.1 in dilute solution (noninteracting rods). The NaGDC moderate concentration (0.10 M) samples with NaCl within the range 0.20-0.60 M are characterized by cylindrical aggregates that, in the presence of repulsive Coulombic interactions progressively more and more screened, produce interference effects, due to the hard-body repulsion and attractive forces, but the agreement between observed and calculated SAXS data is satisfactory. The results of the low and moderate NaGDC concentration samples seem to indicate that the aggregation number increase, produced by adding 0.10 M NaCl, is constant within an ionic strength range and occurs by the addition of oligomers formed by trimers. The samples with a variable NaGDC concentration (0.02-0.10 M) at a fixed and high NaCl concentration (0.6 M) contain cylindrical aggregates that give rise to an attractive term effect prevailing on the hard-body repulsive one. The same situation seems to occur in the NaGDC moderate concentration samples
Study of the complex between the PG of Fusarium phyllophilum and the PGIP2 of Phaseolus vulgaris by intermolecular crosslinking and Small Angular X-Ray Scattering (SAXS)
No full textIbuprofen and propofol cobinding effect on human serum albumin unfolding in urea.
No full textThe 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
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