1,721,019 research outputs found
Denaturation and stabilization of Human Serum Albumin: combined effect of drugs
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). 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 domain open (domain I, which does not host a binding site for either of the ligands), while I2 presents only one closed domain (domain III). A direct comparison with the unfolding pathway of the HSA:Ibu complex 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 which accomodate the ligands. Moreover, the equilibrium between I2 and the Unfolded form is slightly shifted towards higher urea concentrations. These results suggest that the cobinding significantly hinders the unfolding process
Sodium cholate/PEO-PPO-PEO triblock copolymer mixed micelles as stealth nanocarrier for doxorubicin
No full textPolymer nanomaterials have received a great deal of interest as vehicles used for diagnostic and therapeutic agents [1]. The loading efficiency of a bile salt/block copolymer coformulation toward the fluorescent anticancer antibiotic doxorubicin has been studied. The coformulation is based on the anionic bile salt sodium cholate (NaC) and a nonionic triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) denoted EO100-PO65-EO100 (F127) that itself forms micelles in water with a core composed mostly of PPO and a PEO corona. Doxorubicin is usually administered as the hydrochloride (DX) to increase its solubility in water. This limits its partitioning to the corona region of F127 micelles. To promote its solubility in the hydrophobic core, NaC was introduced in the system. The resulting systems obtained by varying the NaC/F127 mole ratio were characterized by small angle X-ray and dynamic light scattering (SAXS and DLS) in combination with spectroscopic fluorescence techniques (steady state and time-resolved). The host structure is not affected by the guest presence as deduced by SAXS and DLS data while in the presence of NaC, DX experiences a more apolar environment as indicated by its characteristic fluorescence behaviour (Figure 1). The stability against degradation of DX in the mixed micellar system was markedly enhanced relative to aqueous solutions without the coformulation [2]. The DX increased time stability in the NaC/pluronic mixed micellar systems is a promising characteristic that could lead to an increase of the drug latency and protection against hydrolytic degradation. In addition, the PEO hydrophilic corona could provide a certain level of biocompatibility and stealth characteristics to the mixed system, thus being attractive from an applicative point of view
Self-Assembly of Model Amphiphilic Peptides in Nonaqueous Solvents : Changing the Driving Force for Aggregation Does Not Change the Fibril Structure
Full text linkWithin 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 β-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 β-sheets and an interfacial free energy gain for the lamination of the hydrophobic β-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 β-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
Structural response of human serum albumin to oxidation: bological buffer to local formation of hypochlorite
Full text linkThe 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 alpha-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
Time-Dependent pH Scanning of the Acid-Induced Unfolding of Human Serum Albumin Reveals Stabilization of the Native Form by Palmitic Acid Binding
No full textThe 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
Structural insights into the shape and assembly of photosynthetic GAPDH/CP12/PRK complex by small angle X-ray scattering
No full textCalvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the scaffold protein CP12, a supramolecular ternary complex of 498 kDa with stoichiometry [(GAPDH)-(CP12)-(PRK)]2. CP12 is an ubiquitous regulatory protein of oxygenic phototrophs that contains, with few exceptions, four conserved cysteines able to form two consecutive disulfide bridges. In higher plants as Arabidopsis thaliana, CP12 is predicted to be an intrinsically disordered protein (IDPs). The activities of GAPDH and PRK enzymes are inhibited by complex formation and fully recovered upon dissociation of the complex at the onset of light, providing an effective means for regulation of the Calvin cycle in vivo. It is proposed that GAPDH/CP12/PRK supramolecular complex occurs in chloroplasts in the dark to ensure strong down-regulation of the Calvin cycle. Thus, the determination of the ternary complex structure is crucial for the understanding of the photosynthetic metabolism in light/dark regime. Crystallization trials to produce single crystals of the complex for X-ray diffraction experiments, failed. A structural study in solution by small angle scattering was then approached.
The scattering profiles of the complex as well as of the PRK dimer were measured on the BM29, the dedicated bioSAXS beamline at the European Synchrotron Radiation Facility (Grenoble) and the ATSAS package was used for data analysis and modeling. First, the ab-initio shape of the PRK dimer was recovered using the program GASBOR. This bent-prolate structural model was then used together with the GAPDH-(CP12)2 complex crystallographic coordinates in the rigid-body modeling of the ternary complex against the experimental scattering curve performed with the program SASREFmx. The known stoichiometry of the complex was confirmed by the optimal data fitting. From the sorting of a big number of models obtained after multiple runs of the minimization procedure, an overall highly reproducible assembly emerged. The two GAPDH tetramers were in close contact and the two PRK dimers, both oriented with the concavity facing the centre of the complex, bridged them by interacting with the GAPDH-bound CP12s through the end regions.
This rigid-body model of the complex was also consistent with previously reported hydrodynamic data. The SAXS-recovered structure is compatible with the present knowledge about this protein complex and highlights the propensity of GAPDH tetramers to interact reciprocally and associate in higher molecular weight forms as already reported from in vitro and in vivo observations
An albumin unfolding and refolding cycle induced by a time-controlled pH jump
Full text linkGiven the intimate connection between the structure and function of biological macromolecules, the ability to temporally control their unfolding-refolding process enables temporal regulation over specific functionalities, potentially applicable in innovative domains, including the construction of protein-based actuators or programmable catalysis and drug release in complex biotechnological processes. We show here how a temporally controlled protein unfolding-refolding cycle can be coupled in time with programmed pH sequences achieved through the spontaneous decomposition of an activated carboxylic acid. Specifically, we illustrate this process at the molecular level using albumin, the most prevalent protein found in plasma, for which a temporary shift from native to unfolded forms is promoted using nitroacetic acid, able to undergo base-catalysed decarboxylation when solubilized in water solution. As detected by small angle X-ray scattering and intrinsic tryptophan fluorescence, starting from the protein in its native form, the acid addition triggers unfolding to a partially denatured state and a subsequent time-tunable pH rise with gradual refolding that recapitulates the intermediate steps detected at the same pH values by static acidification, fitting within a framework of full reversibility of the structural changes as a function of the protein protonation state. At the end of the pH jump, the native structure is fully recovered, making this method a chemical tool to achieve a complete protein conformational sequence programmed in the timeframe of minutes without further intervention.An unfolding-refolding cycle of albumin is accomplished by using a pH jump based on the time-dependent decarboxylation of nitroacetic acid
Anatomy of a deep eutectic solvent: structural properties of choline chloride : sesamol 1 : 3 compared to reline
No full textThe structural properties of the deep eutectic solvent (DES) formed by choline chloride (ChCl) and sesamol in 1 : 3 ratio have been investigated and compared to those of reline (ChCl : urea 1 : 2). An integrated approach combining small and wide angle X-ray scattering with molecular dynamics simulations has been employed and the simulation protocol has been validated against the experimental data. In the ChCl : sesamol DES, strong hydrogen bonds (HBs) are formed between the chloride anion and the hydroxyl groups of the choline and of sesamol molecules. Conversely, choline-choline, choline-sesamol and sesamol-sesamol interactions are negligible. A more extended interplay between the constituents is observed in reline where, besides the HBs involving the chloride anion, the eutectic formation is favored also by strong choline-urea and urea-urea interactions. The three-dimensional arrangement around the individual components shows that, in the ChCl : sesamol DES, the cholinium cations and the sesamol molecules are packed in such a way to maximize the interactions with the chlorine anion. This structural arrangement may favor the pi-pi interactions between the sesamol molecules and the aromatic species mediated by the chloride ions, providing an interpretation for the high separation rates previously observed for phenolic DESs towards aromatic compounds
Insight into the assembly of the Calvin cycle regulatory GAPDH/CP12/PRK complex by SAXS
No full textPhotosynthetic organisms produce sugars through the Calvin-Benson cycle, consuming carbon dioxide and energy provided by the conversion of light to chemical energy. The smooth proceeding of photosynthesis is controlled by different regulatory systems including the transient formation of protein complexes. Through the scaffold protein CP12, which is predicted to be intrinsically disordered, two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (tetrameric GAPDH) and phosphoribulokinase (dimeric PRK), are regulated by formation of a supramolecular ternary complex of 498 kDa with stoichiometry [GAPDH-(CP12)2-(PRK)]2. The activities of GAPDH and PRK enzymes are inhibited by complex formation and fully recovered upon dissociation of the complex at the onset of light, providing an effective means for regulation of the Calvin cycle in vivo.Small angle X-ray scattering analysis was performed on the pre-formed complex and its free components all from Arabidopsis thaliana, and the ATSAS package was used for data analysis and modelling. A concave bent and screwed ab-initio shape of the PRK dimer was recovered, while a combined rigid-body/dummy-residue model was obtained for the GAPDH-(CP12)2 binary complex in order to take into account a small rearrangement of the known crystallographic subunits positions and the missing CP12 amino acids. These models were then used in the rigid-body modelling of the ternary complex against the experimental scattering curve, allowing for partial dissociation. The known stoichiometry of the complex was confirmed and from the sorting of a big number of models obtained with multiple runs of the minimization procedure, an overall reproducible assembly emerged. The structure of the ternary complex appears more compact with respect to the previous pictorial models and the two GAPDHs proximity suggests an unsuspected involvement of an interaction between them in the overall complex stabilization
Ibuprofen 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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