Why has porcine VEG protein unusually high stability and suppressed binding ability?
Von Ebner gland protein (VEGP) and odorant-binding protein (OBP) were purified from porcine lingual epithelium and nasal mucosa, respectively. Both VEGP and OBP preparations were homogeneous as indicated by SDS-PAGE, isoelectric focusing, gel-filtration and electrospray mass spectrometry. However, high-sensitivity differential scanning calorimetry (HS-DSC) yielded multiphasic denaturation thermograms for both proteins indicating their conformational heterogeneity. The unfolding transition of VEGP is observed at extremely high temperatures (about 110 degrees C), which is unexpected for a protein with significant structural homology to OBP and other lipocalins. Isothermal titration calorimetry (ITC) did not detect the binding of either aspartame or denatonium saccharide to VEGP nor did it detect binding of 2-isobutyl-3-methoxypyrazine (IBMP) to OBP. Extraction of OBP with mixed organic solvents eliminated the conformational heterogeneity and the protein showed a reversible two-state transition in HS-DSC thereafter. ITC also showed that the extracted OBP was able to bind IBMP. These results imply that tightly bound endogenous ligands increase the thermal stability of OBP and block the binding of other ligands. In contrast to OBP, the extraction of VEGP with organic solvents failed to promote binding or to establish thermal homogeneity, most likely because of the irreversible denaturation of VEGP. Thus, the elucidation of the functional behaviour of VEGP is closely related to the exhaustive purging of its endogenous ligands which otherwise very efficiently mask ligand binding sites of this protein.
Burova
T V
TV
Institute of Biochemical Physics of the Russian Academy of Sciences, Moscow.
Rabesona
H
H
Choiset
Y
Y
Jankowski
C K
CK
Sawyer
L
L
Haertlé
T
T
D000818:Animals; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004848:Epithelium / Q000737:chemistry; D054835:Lipocalin 1; D009061:Mouth Mucosa / Q000737:chemistry; D009297:Nasal Mucosa / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D011719:Pyrazines / Q000737:chemistry; D018035:Receptors, Odorant / Q000737:chemistry* / Q000302:isolation & purification; D013552:Swine
D002352:Carrier Proteins; D054835:Lipocalin 1; D011719:Pyrazines; D018035:Receptors, Odorant; C045648:odorant-binding protein; C034376:2-isobutyl-3-methoxypyrazine