Effects of i-propanol on the structural dynamics of Thermomyces lanuginosalipase revealed by tryptophan fluorescence

Influence of isopropanol (iPrOH) on the structural dynamics of Thermomyces lanuginosa lipase (TLL) was studied by steady-state, time-resolved, and sto pped-flow fluorescence spectroscopy, monitoring the intrinsic emission of T rp residues. The fluorescence of the four Trps of the wild-type enzyme repo rt on the global changes of the whole lipase molecule. To monitor the confo rmational changes in the so-called "lid," an or-helical surface loop, the s ingle Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichrois m (CD) spectra revealed that iPrOH does not cause major alterations in the secondary structures of the wild-type TLL and W89m. With increasing [iPrOH] , judged by the ratio of emission intensities at 350 nm and 330 nm, the ave rage microenvironment of the Trps in the wild-type TLL became more hydropho bic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time-resolved fluorescence measurements revealed no major changes to be ind uced by iPrOH neither in the shorter fluorescence lifetime component (tau ( 1) = 0.5-1.2 ns) for the wild-type TLL nor in the longer fluorescence lifet ime component (tau (2) = 4.8-6.0 ns) in the wild-type TLL and the W89m muta nt. Instead, for W89m on increasing iPrOH from 25% to 50% the value for tau (1) increased significantly, from 0.43 to 1.5 ns. The shorter correlation time phi (1) of W89m had a minimum of 0.08 ns in 25% iPrOH. Judged from the residual anisotropy r(infinity) the amplitude of the local motion of Trp89 increased upon increasing [iPrOH] 10%. Stopped-flow fluorescence spectrosc opy measurements suggested the lid to open within approximate to 2 ms upon transfer of W89m into 25% iPrOH. Steady-state anisotropies and longer corre lation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.