Cutinase unfolding and stabilization by trehalose and mannosylglycerate

The unfolding of cutinase at pH 4.5 was induced by increasing the temperatu re and guanidine hydrochloride concentration in the presence of potassium c hloride, trehalose, and mannosyl-glycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions w ere coincident within experimental error when assessed by near-ultraviolet (UV) difference, tryptophyl, and 8-anilino-1-naphthalene sulfonic acid (ANS ) fluorescence spectroscopy. Trehalose at 0.5 M increased the temperature a t which 50% of cutinase is unfolded by 3 degreesC. Unfolding induced by gua nidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV diff erence spectroscopy occurs earlier than unfolding assessed by tryptophyl fl uorescence. The intermediate is molten globule in character: the ANS fluore scence is higher than in the presence of the folded or unfolded state, show ing native-like secondary structure and losing many tertiary interactions o f the folded state, i.e., those surrounding the tyrosyl microenvironment. T he stabilization effect of trehalose and mannosylglycerate was quantified b y fitting the unfolding transitions to a model proposed by Staniforth et al , (Biochemistry 1993;32: 3842-3851). This model takes into consideration th e increase in solvation energies of the amino acid side-chains as the denat urant concentration was increased and the fraction of amino acid side-chain s that become exposed in the unfolded structure of cutinase, Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate b y 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded st ate by 1.0 and 1.5 kcal/mol, respectively. (C) 2001 Wiley-Liss, Inc.