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

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractSmall 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 SingularValue 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 I1(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 & 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.

Structural Resolution of the Complex between a Fungal Polygalacturonase and a Plant Polygalacturonase-Inhibiting Protein by Small-Angle X-Ray Scattering

“…By using ab initio and rigid body modeling (Svergun, 1999;Chacón et al, 2000;Heller et al, 2003), the shape of monomeric forms, the multimeric state of proteins, and protein complexes can be assessed (Putnam et al, 2007;Mertens and Svergun, 2010;Nogales et al, 2010;Pons et al, 2010). In addi- tion, SAXS allows the quantitative analysis of flexible systems including multidomain and intrinsically unfolded proteins (Bernadó et al, 2005), mechanisms of denaturation and unfolding (Segel et al, 1998;Galantini et al, 2008;Leggio et al, 2009), the stabilizing role of the ligands in denaturating conditions (Galantini et al, 2010), and molecular mechanisms of ligand release (Tabarani et al, 2009).…”

Structural analysis and binding domain of albumin complexes with natural dietary supplement humic acid

“…The inside wall of the pocket is formed by hydrophobic side chains, whereas the entrance to the cavity is surrounded by positively charged residues. The exceptional feature of this site is the binding of the ligand, which is a bulky heterocyclic anion with a negative charge localized in the middle of the molecule [48]. Ligands binding in site I include warfarin, phenylbutazone, and azapropazone.…”

Histidine146 of human serum albumin plays a prominent role at the interface of subdomains IA and IIA in allosteric ligand binding

“…In an investigation of the stabilizing effect of ibuprofen in the unfolding of HSA induced by urea, Galantini et al reported that domain I, which contains polar residues, is capable of participating in electrostatic interactions with ibuprofen, an arylpropionic acid nonsteroidal anti-inflammatory drug that is similar to KP (26). Hence, in this study, we attempted to clarify the mechanism for the effect of allosteric binding from the perspective of an N-B transition.…”