Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate.
The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the alpha-helical surface loop, the so-called 'lid' of HLL comprised of residues 86-93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the 'open' conformation in triacetin, with the alpha-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.
Jutila
Arimatti
A
Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, PO Box 63 (Haartmaninkatu 8), FIN-00014 University of Helsinki, Finland. arimatti.jutila@helsinki.fi
Zhu
Keng
K
Tuominen
Esa K J
EK
Kinnunen
Paavo K J
PK
D016880:Anisotropy; D000935:Antifungal Agents / Q000737:chemistry* / Q000378:metabolism; D001203:Ascomycota / Q000201:enzymology*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014215:Triacetin / Q000737:chemistry* / Q000378:metabolism
D000935:Antifungal Agents; D005656:Fungal Proteins; D008049:Lipase; D014215:Triacetin