Steady state and time resolved effects of guanidine hydrochloride on the structure of Humicola lanuginosa lipase revealed by fluorescence spectroscopy

Effects of guanidine hydrochloride (GdnHCl) on the structure and dynamics o f wild-type Humicola lanuginosa lipase (HLL) and its two mutants were studi ed. The latter were S146A (with the active site Ser replaced by Ala) and th e single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Stead y-state, stopped-flow, and time-resolved laser-induced fluorescence spectro scopy were carried out as a function of [GdnHCl]. The maximum emission wave length and fluorescence lifetimes revealed the microenvironment of the tryp tophan(s) in these lipases to become more polar upon increasing [GdnHCl]. H owever, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild-type HLL and W89m remain catalytically activ e at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Chang es in steady-state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon inc reasing [GdnHCl] the structure of the lipases became more loose, with incre asing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped- flow fluorescence measurements.