Unfolding and refolding of porcine odorant binding protein in guanidinium hydrochloride: equilibrium studies at neutral pH.
Unfolding and refolding studies on porcine odorant binding protein (pOBP) have been performed at pH 7 in the presence of guanidinium hydrochloride (GdnHCl). Unfolding, monitored by following changes of protein fluorescence and circular dichroism (CD), was found to be a reversible process, in terms of recovered structure and function. The equilibrium transition data were fitted by a simple two-state sigmoidal function of denaturant concentration and the thermodynamic folding parameters, derived from the two techniques, were very similar (average values: C(1/2) approximately 2.4 M, m approximately 2 kcal mol(-1) M(-1), DeltaG(unf,w)(0) approximately 4.7 kcal mol(-1)). The transition was independent of protein concentration, indicating that only monomeric species are involved. Only a minor protective effect by the fluorescent ligand 1-amino-anthracene (AMA) against protein unfolding was detected, whereas dihydromyrcenol (DHM) stabilised the protein to a larger extent (DeltaC(1/2) approximately 0.5 M). Refolding was complete, when the protein, denatured with GdnHCl, was diluted with buffer. On the other hand, refolding by dialysis was largely prevented by concomitant aggregation. The present results on pOBP are compared with those on bovine OBP (bOBP) [Biochim. Biophys. Acta 1599 (2002) 90], where subunit folding is accompanied by domain swapping. We finally suggest that the generally observed two-state folding of many lipocalins is probably favoured by their beta-barrel topology.
Parisi
Mariella
M
Istituto Nazionale di Fisica della Materia (INFM), Unità di Parma, Università di Parma, Parma, Italy.
Mazzini
Alberto
A
Sorbi
Robert T
RT
Ramoni
Roberto
R
Grolli
Stefano
S
Favilla
Roberto
R
D000818:Animals; D000873:Anthracenes / Q000378:metabolism; D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D018035:Receptors, Odorant / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013552:Swine
D000873:Anthracenes; D018035:Receptors, Odorant; C045648:odorant-binding protein; C049062:1-aminoanthracene; D019791:Guanidine