Effects of removing a conserved disulfide bond on the biological characteristics of rat lipocalin-type prostaglandin D synthase.
Three cysteine residues, Cys(65), Cys(89), and Cys(186) in lipocalin-type prostaglandin D synthase (L-PGDS), are conserved among all species and the disulfide bond between Cys(89) and Cys(186) is highly conserved among most, but not all, lipocalins. In this study, four rat L-PGDS variants were constructed by site-directed mutagenesis, and the conserved disulfide bond in several variants was removed by substituting cysteine with alanine. The effects of removing this disulfide bond on their biological characteristics were investigated. The NMR experiments indicated that the removal of disulfide did not change their conformations significantly. However, both thermal-induced and urea-induced unfolding experiments showed that the stabilities of enzymes without the disulfide bond decreased significantly. Moreover, the ligand-binding affinities of these variants were assessed by fluorescence experiments. Dissociation constants (K(d)) of 0.668, 0.689, 0.543 and 0.571 microM were obtained for ANS binding to wild-type rat L-PGDS, C(65)A, C(186)A, and C(89,186)A variants, respectively, and 71.2 and 62.3 nM for retinoic acid binding to wild-type rat L-PGDS and the C(186)A variant, respectively. These results suggested that the removal of the disulfide bond slightly increased the affinities for ligand binding by changing the hydrophobic regions. This study may offer valuable information for further studies on other rat lipocalins.
Liu
Jiafu
J
School of Life Science and Technology, China Pharmaceutical University, Nanjing, China. jiafuliu@gmail.com
Guo
Chenyun
C
Yao
Yihe
Y
Lin
Donghai
D
D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054834:Lipocalins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
D004220:Disulfides; D054834:Lipocalins; D011994:Recombinant Proteins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D003545:Cysteine