Fluorescence study of fungal lipase from Humicola lanuginosa

Abstract

Time-resolved and steady-state fluorescence quenching measurements have been performed to study two different conformations of the fungal lipase from Humicola lanuginosa. The intrinsic fluorescence of tryptophan Trp89 residue, located in the ‘lid’ region, has been used as a probe for the dynamics of protein. The native (‘closed-lid’) form of the enzyme has been found to decay as a triple exponential with time constants and relative contributions of 5.4 ns (74.3%), 2.2 ns (20.4%) and 0.4 ns (5.3%). A comparison of recovered decay parameters obtained for native and mutated H. lanuginosa lipase shows that Trp89 contributes about 61% to the class of emitting species with the lifetime of 5.4 ns. The fluorescence quenching data show that three out of four tryptophans (i.e., 117, 221 and 260 residues) with H. lanuginosa lipase are totally quenchable by acrylamide while completely inaccessible to iodide. On the contrary, the Trp89 residue is available for both quenchers. Using steady-state iodide fluorescence quenching data and the fluorescence-quenching-resolved-spectra (FQRS) method, the total emission spectrum of the native lipase has been decomposed into two spectral components. One of them, unquenchable by iodide, has a maximum of fluorescence emission at 330 nm and the second one, exposed to the solvent, emits at 338 nm. The resolved spectrum of the redder component corresponds to the Trp89 residue, which participates in about 65% of the total H. lanuginosa emission. The dynamic Stern—Volmer quenching constants calculated for both native (‘closed-lid’) and inhibited (‘open-lid’) lipase are 2.71 and 4.49 M −1, respectively. The values obtained indicate that Trp89 is not deeply buried in the protein matrix. Our results suggest that distinct configurations of fungal lipase can be monitored using the fluorescence of the Trp89 residue located in the ‘lid’-helix which participates in an interfacial activation of the enzyme.