title: Native disulfide bonds in plasma retinol-binding protein are not essential for all-trans-retinol-binding activity.
abstract: A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.
authors.affiliation: Center for Advanced Biotechnology, Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Gabriel O
authors.initials: GO
authors.lastname: Reznik
authors.forename: Yong
authors.initials: Y
authors.lastname: Yu
authors.forename: George E
authors.initials: GE
authors.lastname: Tarr
authors.forename: Charles R
authors.initials: CR
authors.lastname: Cantor
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D012177:Retinol-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D054839:Retinol-Binding Proteins, Plasma; D013816:Thermodynamics; D014801:Vitamin A / Q000378:metabolism*
chemical_list: D004220:Disulfides; D008024:Ligands; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma; D014801:Vitamin A