Volume 78, Issue 3, Pages (March 2000)

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Volume 78, Issue 3, Pages 1634-1642 (March 2000) Detergent-Induced Conformational Changes of Humicola lanuginosa Lipase Studied by Fluorescence Spectroscopy Arimatti Jutila, Keng Zhu, Shamkant Anant Patkar, Jesper Vind, Allan Svendsen, Paavo K.J. Kinnunen Biophysical Journal Volume 78, Issue 3, Pages 1634-1642 (March 2000) DOI: 10.1016/S0006-3495(00)76715-4 Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 1 Fluorescence emission spectra of free tryptophan (·····) and wild-type (——) and W89m HLL (– – –). The spectra were normalized to the same maximum value for emission intensity. The peak intensity values corrected with respect to Trp concentrations were 100, 70, and 130 for free Trp, wt, and W89m. The excitation wavelength was 295nm. The concentrations of Trp and the lipases were 1μM in 20mM Hepes, 0.1mM EDTA (pH 7.0). The temperature was 25°C. Biophysical Journal 2000 78, 1634-1642DOI: (10.1016/S0006-3495(00)76715-4) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 2 Quenching of tryptophan emission by KI in wt (●) and W89m (■). The relative fluorescence intensity values in the absence of KI are normalized to 100. The conditions were as described in Experimental Procedures. Biophysical Journal 2000 78, 1634-1642DOI: (10.1016/S0006-3495(00)76715-4) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 3 (A) Relative fluorescence intensity of free tryptophan (□) and wild-type (●) and mutant (■) HLL as a function of [C8E5]. (B) Emission intensity values in the absence of C8E5 are normalized to 100. The conditions used were identical to those described in the legend for Fig. 1. Biophysical Journal 2000 78, 1634-1642DOI: (10.1016/S0006-3495(00)76715-4) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 4 Steady-state fluorescence anisotropy of free tryptophan (□) and wild-type (●) and mutant (■) HLL as a function of [C8E5], measured at 25°C in 20mM Hepes, 0.1mM EDTA (pH 7.0). The concentration of Trp and lipases was 1μM. Biophysical Journal 2000 78, 1634-1642DOI: (10.1016/S0006-3495(00)76715-4) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 5 Fluorescence lifetimes τ1 (A) and τ2 (B) and the fractional intensity of the component with a lifetime τ2 (C) of free tryptophan (□) and wild-type (●) and mutant (■) HLL at different [C8E5]. Conditions were as stated in the legend for Fig. 1. Biophysical Journal 2000 78, 1634-1642DOI: (10.1016/S0006-3495(00)76715-4) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 6 Rotational correlation times ϕ1 (A) and ϕ2 (B) and the residual anisotropy r∞ (C) of W89m HLL at different [C8E5], measured as described in Experimental Procedures. The temperature was 25°C. Biophysical Journal 2000 78, 1634-1642DOI: (10.1016/S0006-3495(00)76715-4) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 7 Relative activity of wt (●) and W89m (■) HLL toward PNPB at different C8E5 concentrations, as indicated by the change in absorbance at 405nm. The concentrations of HLL and PNPB were 0.33μM and 1mM in 20mM HEPES, 0.1mM EDTA (pH 7.0). The temperature was 25°C. Biophysical Journal 2000 78, 1634-1642DOI: (10.1016/S0006-3495(00)76715-4) Copyright © 2000 The Biophysical Society Terms and Conditions