Role of the disulphide bridge in folding, stability and function of porcine odorant binding protein: spectroscopic equilibrium studies on C63A/C155A double mutant.
Porcine odorant binding protein (pOBP) contains a single disulphide bridge linking residues Cys63 and Cys155. In order to get information on the role played by this crosslink in determining the structural and functional properties of the protein, we substituted these two Cys residues with two Ala residues by site directed mutagenesis and investigated the changes in folding, stability and functional features, as detected by fluorescence and circular dichroism measurements. In particular, we studied both chemical and thermal unfolding/refolding processes under equilibrium conditions, the first induced by guanidinium hydrochloride and the second by raising the temperature from 15 to 90 degrees C. Chemical unfolding curves, as obtained from intrinsic fluorescence and far-UV circular dichroism data, can be fitted by a simple two-state cooperative sigmoidal function; however, their partial overlap (C(1/2)=0.57+/-0.05 from fluorescence and 0.66+/-0.03 from CD) suggests the formation of an intermediate, which lacks tertiary structural features. Thermal unfolding was found to be reversible if the protein was heated up to 65 degrees C, but irreversible above that temperature because of aggregation. The thermodynamic unfolding parameters of this double mutant protein, when compared to those of the wild type protein, clearly point out the important role played by the disulphide bridge on the stability and function of this protein family and probably of many other lipocalins.
Parisi
Mariella
M
Istituto Nazionale di Fisica della Materia (INFM)-Unità di Parma, Italy.
Mazzini
Alberto
A
Tibor Sorbi
Robert
R
Ramoni
Roberto
R
Grolli
Stefano
S
Favilla
Roberto
R
D000818:Animals; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D008024:Ligands; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D018035:Receptors, Odorant / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013552:Swine; D013816:Thermodynamics
D004220:Disulfides; D008024:Ligands; D018035:Receptors, Odorant; C045648:odorant-binding protein; D019791:Guanidine; D003545:Cysteine