Thermodynamics and mechanism of cutinase stabilization by trehalose

Abstract

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy-enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding.

Co-solvent mediated thermal stabilization of chondroitinase ABC I form Proteus vulgaris

“…Studies have also demonstrated that surface tension of trehalose solutions depends on trehalose concentration and there is a direct correlation between surface tension and T m [39]. The present study demonstrates a significant decrease in rcABC I flexibility in the presence of trehalose which has been reported for numerous enzymes [36,37,40,41]. Since trehalose is approved by FDA as a safe cosolvent, it can be used as a special stabilizer for cABC I stabilization and be administered in vivo in treatment of SCI.…”

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential

“…Compared to its close homologue, originating from F. solani, it seems slightly more thermotolerant, as it retains 80% of its activity after 1h incubation at 30 o C, compared to 60% in the case of F. solani cutinase [28]. Improving the robustness of the latter has been the target of intensive research efforts, due to its potential as industrial biocatalyst [12,49,69] and a Z-score of 10.9 (Dali server). A structure-based sequence alignment between the two proteins did not show any conservation of the residues implicated in calcium binding.…”

Effect of trehalose on protein structure

“…96 In case of unfolding of cutinase by heat, trehalose increases the melting temperature of the enzyme by delaying thermal unfolding of an intermediate. 97 Trehalose compensates for the altered entropy of the process by increasing the contribution of enthalpy to the process, making unfolding a thermodynamically unfavorable process. On exposure to heat, yeast produces trehalose, along with heat shock proteins, on a large scale.…”