Structural effects resulting from the interaction of the anionic surfactant sodium bis[2-ethylhexyl]ester sulfosuccinic acid (AOT) with a recombinant cutinase are studied and characterised by means of spectroscopic techniques. Levels of interaction are described in terms of surfactant to protein molar ratio (MR). Three major regions may be identified: MR = 0-10, MR = 10-30 and MR > 30. The latter corresponds to co-operative binding of the surfactant to protein leading to overall denaturation as observed by far-UV circular dichroism (CD) and 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) fluorescence. For MR = 10-30, steady-state fluorescence suggests slight conformational changes while near-UV CD shows almost complete loss of signal for the chromophore residues. Finally, the first level, MR = 0-10, reveals two distinct effects of interaction. For very low MR (0-5), the protein seems to remain structurally intact. However, at MR = 10, both near-UV CD and unfolding kinetics reveal a structurally disturbed protein contrary to steady-state fluorescence spectra. This suggests that AOT interacts specifically with cutinase at this level, through electrostatic interactions mostly. By promoting localised disruption or destabilisation of crucial native electrostatic interactions, the surfactant initiates conformational loss of tertiary structure, leading to higher denaturation as MR increases. (C) 2003 Elsevier Science Inc. All rights reserved.