Effects of i-propanol on the structural dynamics of Thermomyces lanuginosa lipase 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 stopped-flow fluorescence spectroscopy, monitoring the intrinsic emission of Trp residues. The fluorescence of the four Trps of the wild-type enzyme report on the global changes of the whole lipase molecule. To monitor the conformational changes in the so-called "lid," an alpha-helical surface loop, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Circular dichroism (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 average microenvironment of the Trps in the wild-type TLL became more hydrophobic, whereas Trp89 of W89m moved into a more hydrophilic microenvironment. Time-resolved fluorescence measurements revealed no major changes to be induced 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 lifetime component (tau(2) = 4.8--6.0 ns) in the wild-type TLL and the W89m mutant. 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 spectroscopy measurements suggested the lid to open within approximately 2 ms upon transfer of W89m into 25% iPrOH. Steady-state anisotropies and longer correlation times revealed increasing concentrations of iPrOH to result also in the formation of dimers as well as possibly also higher oligomers by TLL.
Helsinki Biophysics and Biomembrane Group, Department of Medical Chemistry, Institute of Biomedicine, FIN-00014 University of Helsinki, Finland.
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D019840:2-Propanol / Q000737:chemistry* / Q000494:pharmacology; D016880:Anisotropy; D001203:Ascomycota / Q000737:chemistry*; D002384:Catalysis; D002942:Circular Dichroism; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D015233:Models, Statistical; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*; D014466:Ultraviolet Rays
D014364:Tryptophan; D008049:Lipase; D019840:2-Propanol