Tryptophan to phenylalanine substitutions allow differentiation of short- and long-range conformational changes during denaturation of goat α-lactalbumin

Ann Vanhooren, Allel Chedad, Viktor Farkas, Zs. Majer, Marcel Joniau, Herman Van Dael, Ignace Hanssens

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

To test the occurrence of local particularities during the unfolding of Ca2+-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 (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (T m,Fl int) and from DSC (Tm,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.

Original languageEnglish
Pages (from-to)118-130
Number of pages13
JournalProteins: Structure, Function and Genetics
Volume60
Issue number1
DOIs
Publication statusPublished - Jun 15 2005

Fingerprint

Lactalbumin
Denaturation
Phenylalanine
Goats
Tryptophan
Substitution reactions
Fluorescence
Transition Temperature
Quenching
Hot Temperature
Value engineering
Pichia
Methionine
Free energy
Conformations
Kinetics
Temperature

Keywords

  • Chemical unfolding
  • Circular dichroism
  • Differential scanning calorimetry
  • Fluorescence
  • Protein folding
  • Stopped-flow kinetics
  • Thermal unfolding
  • Trp-Phe mutants

ASJC Scopus subject areas

  • Genetics
  • Structural Biology
  • Biochemistry

Cite this

Tryptophan to phenylalanine substitutions allow differentiation of short- and long-range conformational changes during denaturation of goat α-lactalbumin. / Vanhooren, Ann; Chedad, Allel; Farkas, Viktor; Majer, Zs.; Joniau, Marcel; Van Dael, Herman; Hanssens, Ignace.

In: Proteins: Structure, Function and Genetics, Vol. 60, No. 1, 15.06.2005, p. 118-130.

Research output: Contribution to journalArticle

Vanhooren, Ann ; Chedad, Allel ; Farkas, Viktor ; Majer, Zs. ; Joniau, Marcel ; Van Dael, Herman ; Hanssens, Ignace. / Tryptophan to phenylalanine substitutions allow differentiation of short- and long-range conformational changes during denaturation of goat α-lactalbumin. In: Proteins: Structure, Function and Genetics. 2005 ; Vol. 60, No. 1. pp. 118-130.
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AU - Vanhooren, Ann

AU - Chedad, Allel

AU - Farkas, Viktor

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AU - Joniau, Marcel

AU - Van Dael, Herman

AU - Hanssens, Ignace

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AB - To test the occurrence of local particularities during the unfolding of Ca2+-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 (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (T m,Fl int) and from DSC (Tm,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.

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