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Tryptophan to phenylalanine substitutions allow differentiation of short‐ and long‐range conformational changes during denaturation of goat α‐lactalbumin
Author(s) -
Vanhooren Ann,
Chedad Allel,
Farkas Viktor,
Majer Zsuzsa,
Joniau Marcel,
Van Dael Herman,
Hanssens Ignace
Publication year - 2005
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20496
Subject(s) - chemistry , quenching (fluorescence) , fluorescence , denaturation (fissile materials) , crystallography , lactalbumin , tryptophan , mutant , circular dichroism , biochemistry , amino acid , nuclear chemistry , physics , quantum mechanics , gene
Abstract To test the occurrence of local particularities during the unfolding of Ca 2+ ‐loaded goat α‐lactalbumin (GLA) we replaced Trp60 and ‐118, either one or both, by Phe. In contrast with alternative studies, our recombinant α‐lactalbumins are expressed in Pichia pastoris and do not contain the extra N‐terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and ‐118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and ‐104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima ( T m ,Fl λ ) and those derived from DSC ( T m ,DSC ). However, the transition temperatures derived from fluorescence intensity ( T m ,Fl int ) and from DSC ( T m ,DSC ), respectively, are quite similar, and thus, no local rearrangements are observed upon heat‐induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl‐induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped‐flow fluorescence measurements. By ϕ ‡ ‐value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native. Proteins 2005. © 2005 Wiley‐Liss, Inc.