title: Dissociation and unfolding of bovine odorant binding protein at acidic pH.
abstract: Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.
authors.affiliation: Dipartimento di Fisica and CNISM, Università di Parma, Viale Usberti, 7A, 43100 Parma, Italy. alberto.mazzini@fis.unipr.it
authors.forename: Alberto
authors.initials: A
authors.lastname: Mazzini
authors.forename: Eugenia
authors.initials: E
authors.lastname: Polverini
authors.forename: Mariella
authors.initials: M
authors.lastname: Parisi
authors.forename: Robert Tibor
authors.initials: RT
authors.lastname: Sorbi
authors.forename: Roberto
authors.initials: R
authors.lastname: Favilla
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D002417:Cattle; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D018035:Receptors, Odorant / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D018035:Receptors, Odorant; C045648:odorant-binding protein