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

Published online by Cambridge University Press:  01 March 2000

KENG ZHU ,
ARIMATTI JUTILA  and
PAAVO K.J. KINNUNEN
KENG ZHU
Affiliation:
University of Helsinki, Institute of Biomedicine, Department of Medical Chemistry, Siltavuorenpenger 10A, Helsinki FIN-00014, Finland
ARIMATTI JUTILA
Affiliation:
University of Helsinki, Institute of Biomedicine, Department of Medical Chemistry, Siltavuorenpenger 10A, Helsinki FIN-00014, Finland
PAAVO K.J. KINNUNEN
Affiliation:
University of Helsinki, Institute of Biomedicine, Department of Medical Chemistry, Siltavuorenpenger 10A, Helsinki FIN-00014, Finland
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Abstract

Effects of guanidine hydrochloride (GdnHCl) on the structure and dynamics of wild-type Humicola lanuginosa lipase (HLL) and its two mutants were studied. The latter were S146A (with the active site Ser replaced by Ala) and the single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Steady-state, stopped-flow, and time-resolved laser-induced fluorescence spectroscopy were carried out as a function of [GdnHCl]. The maximum emission wavelength and fluorescence lifetimes revealed the microenvironment of the tryptophan(s) in these lipases to become more polar upon increasing [GdnHCl]. However, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild-type HLL and W89m remain catalytically active at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Changes in steady-state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon increasing [GdnHCl] the structure of the lipases became more loose, with increasing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped-flow fluorescence measurements.

Keywords

fluorescence spectroscopyHumicola lanuginosa lipaseunfolding
Type
Research Article
Information
Protein Science , Volume 9 , Issue 3 , March 2000 , pp. 598 - 609
Copyright
© 2000 The Protein Society

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