title: Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin.
abstract: Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0M TMAO increased the free energy change (ΔG(0)) significantly from 2.1 to 3.8kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.
authors.affiliation: Departments of Pathology and Ophthalmology and Jules Stein Eye Institute, University California at Los Angeles, CA 90095, United States. Electronic address: ogassymov@mednet.ucla.edu.
authors.forename: Oktay K
authors.initials: OK
authors.lastname: Gasymov
authors.affiliation: Departments of Pathology and Ophthalmology and Jules Stein Eye Institute, University California at Los Angeles, CA 90095, United States.
authors.forename: Adil R
authors.initials: AR
authors.lastname: Abduragimov
authors.affiliation: Departments of Pathology and Ophthalmology and Jules Stein Eye Institute, University California at Los Angeles, CA 90095, United States. Electronic address: bglasgow@mednet.ucla.edu.
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Glasgow
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D054834:Lipocalins / Q000737:chemistry* / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013666:Tears / Q000737:chemistry*
chemical_list: D004220:Disulfides; D054834:Lipocalins
keywords: Disulfide bond; Ligand binding kinetics; Protein destabilization; TMAO; Urea unfolding