Cutinase unfolding and stabilization by trehalose and mannosylglycerate.
The unfolding of cutinase at pH 4.5 was induced by increasing the temperature and guanidine hydrochloride concentration in the presence of potassium chloride, trehalose, and mannosylglycerate potassium salt. Protein thermal unfolding approached a two-state process, since the unfolding transitions were 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 at which 50% of cutinase is unfolded by 3 degrees C. Unfolding induced by guanidine hydrochloride is clearly a non-two-state process. The presence of a stable intermediate was detected because unfolding assessed by near-UV difference spectroscopy occurs earlier than unfolding assessed by tryptophyl fluorescence. The intermediate is molten globule in character: the ANS fluorescence is higher than in the presence of the folded or unfolded state, showing native-like secondary structure and losing many tertiary interactions of the folded state, i.e., those surrounding the tyrosyl microenvironment. The stabilization effect of trehalose and mannosylglycerate was quantified by fitting the unfolding transitions to a model proposed by Staniforth et al. (Biochemistry 1993;32:3842-3851). This model takes into consideration the increase in solvation energies of the amino acid side-chains as the denaturant concentration was increased and the fraction of amino acid side-chains that become exposed in the unfolded structure of cutinase. Trehalose and mannosylglycerate stabilize the folded state relative to the intermediate by 1.4-1.6 and 1.6 kcal/mol and the intermediate relative to the unfolded state by 1.0 and 1.5 kcal/mol, respectively.
Melo
E P
EP
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. emelo@ualg.pt
Faria
T Q
TQ
Martins
L O
LO
Gonçalves
A M
AM
Cabral
J M
JM
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D005453:Fluorescence; D005988:Glyceric Acids / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008358:Mannose / Q000031:analogs & derivatives* / Q000494:pharmacology*; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014199:Trehalose / Q000494:pharmacology*
D005988:Glyceric Acids; C402688:mannosylglycerate; D014199:Trehalose; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D008358:Mannose