Stabilizing osmolytes’ effects on the structure, stability and function of tc-tenecteplase: A one peptide bond digested form of tenecteplase

Organic osmolytes as chemical chaperones, are major cellular compounds that cause protein stabilization in the native form. In the present study, the possible chaperone effects of the three naturally occurring osmolytes (mannitol, sucrose, and trehalose) on the two-chain form of tenecteplase (tc-TNK), a recombinant, genetically engineered mutant tissue plasminogen activator, have been examined using circular dichroism, steady-state fluorescence, UV-Visible spectroscopy, and in silico experiments. The two-chain form is derived from the single-chain protein upon disruption of one peptide bond. We observed that the protein is stable up to a temperature of 64°C. Besides, it folds back to the native conformation from an unfolded state at the temperature as high as 80 °C. Monitoring change in absorption on unfolding and refolding gave a one-step thermal denaturation curve. Thermal denaturation experiments showed a slight more stabilizing effects of the three co-solvents on two-chain in comparison to that on single-chain form. Unlike single-chain tenecteplase, the two-chain form undergoes reversible denaturation which is somehow perturbed in some cases as the result of the presence of osmolytes. Very minor changes in the secondary structure and the tertiary structure were observed. The molecular dynamics simulations and comparative structural analysis of catalytic domain of the protein in the single-chain and two-chain forms in pure water, mannitol/water, trehalose/water, and sucrose/water showed that while the stabilizing effect of the three osmolytes on tc-TNK is induced by preferential accumulation of these molecules around the nonpolar and aromatic residues (i.e., osmolytes decrease the interactions of water molecules with the hydrophobic residues of tc-TNK), the single-chain form is stabilized by preferential exclusion effect.

کلیدواژه ها:

Chemical chaperone ، Two-chain tenecteplase (tc-TNK) ، Single-chain tenecteplase (sc-TNK) ، Tissue plasminogen activator (t-PA) ، Thermal unfolding ، MD simulation