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 thrimethylamine 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, 1.0 M TMAO increased the free energy change (ΔG0) significantly from 2.1 to 3.8 kcal/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.

Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies

“…In a comparison of wild-type protein and single point mutation (I188K), it has been observed that disulfide bond mutation in I188K/S19C/G24C had a better stability and the activity of protein remained longer (Fig. 3), indicating that the disulfide bond played an important role in the stability of protein [10, 18, 22, 30]. I188K/S19C/G24C was more stable than A138E/R50C/Q147C and S190Y/E183C/I188C at 4 °C, 25 °C and 37 °C (Fig.…”

Oxidation Protection in Metal-Binding Peptide Motif and Its Application to Antibody for Site-Selective Conjugation

“…However, the presence of intrinsic cysteine residues in some proteins complicates site-selective conjugation if those residues are not the ideal conjugation target. Moreover, intrinsic cysteine residues are usually present in disulfide bonds, sustaining the protein’s conformational stability [ 9 , 10 ]. Therefore, labeling a fluorophore or other hydrophobic moiety at these positions might compromise overall protein stability, and conjugation with a small molecule can significantly decrease the enzymatic activity [ 7 , 11 ].…”