Reversible unfolding of bovine odorant binding protein induced by guanidinium hydrochloride at neutral pH.
An analysis of the unfolding and refolding curves at equilibrium of dimeric bovine odorant binding protein (bOBP) has been performed. Unfolding induced by guanidinium chloride (GdnHCl) is completely reversible as far as structure and ligand binding capacity are concerned. The transition curves, as obtained by fluorescence and ellipticity measurements, are very similar and have the same protein concentration-independent midpoint (C1/2 approximately 2.6 M). This result implies a sequential, rather than a concerted, unfolding mechanism, with the involvement of an intermediate. However, since it has not been detected, this intermediate must be present in small amounts or have the same optical properties of either native or denatured protein. The thermodynamic best fit parameters, obtained according to a simple two-state model, are: deltaG degrees un,w = 5.0 +/- 0.6 kcal mol(-1), m = 1.9 +/- 0.2 kcal mol(-1) M(-1) and C1/2 = 2.6 +/- 0.1 M. The presence of the ligand dihydromyrcenol has a stabilising effect against unfolding by GdnHCl, with an extrapolated deltaG degrees un,w of 22.2 +/- 0.9 kcal mol(-1), a cooperative index of 3.2 +/- 0.3 and a midpoint of 4.6 +/- 0.4 M. The refolding curves, recorded after 24 h from dilution of denaturant are not yet at equilibrium: they show an apparently lower midpoint (C1/2 = 2.2 M), but tend to overlap the unfolding curve after several days. In contrast to chromatographic unfolding data, which fail to reveal the presence of folded intermediates, chromatographic refolding data as a function of time clearly show a rapid formation of folded monomers, followed by a slower step leading to folded dimers. Therefore, according to this result, we believe that the preferential unfolding/refolding mechanism is one in which dimer dissociation occurs before unfolding rather than the reverse.
Mazzini
Alberto
A
Istituto Nazionale di Fisica della Materia, Unità di Parma, Italy. alberto.mazzini@fis.unipr.it
Maia
Alessia
A
Parisi
Mariella
M
Sorbi
Robert Tibor
RT
Ramoni
Roberto
R
Grolli
Stefano
S
Favilla
Roberto
R
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D018035:Receptors, Odorant / Q000737:chemistry / Q000187:drug effects*
D018035:Receptors, Odorant; C045648:odorant-binding protein; D019791:Guanidine