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Ligand-induced conformation change in folate-binding protein
N C Kaarsholm1, A M Kolstrup, S E Danielsen
1Novo Research Institute, Novo Nordisk A/S, Bagsvaerd, Denmark.
The Biochemical Journal
|June 15, 1993
Summary
Investigating cow
Area of Science:
- Biochemistry
- Structural Biology
- Spectroscopy
Background:
- Folate-binding protein (FBP) from cow's whey plays a crucial role in folate transport.
- Understanding ligand binding effects on FBP structure and stability is essential for its functional characterization.
Purpose of the Study:
- To investigate the impact of folate binding on the secondary structure and stability of cow's whey FBP.
- To characterize the folding and unfolding pathways of FBP using biophysical techniques.
Main Methods:
- Circular dichroism (C.D.) spectroscopy to determine secondary structure and monitor unfolding.
- Fluorescence spectroscopy to assess tryptophan fluorescence quenching upon ligand binding.
- Guanidinium chloride-induced denaturation to study protein unfolding.
Main Results:
- Unligated FBP secondary structure predicted as 22% helix, 25% antiparallel beta-strand, 5% parallel beta-strand, 17% turn, and 31% random coil.
- Folate binding induced a 10% decrease in antiparallel beta-strand content and strong quenching of tryptophan fluorescence.
- FBP unfolding is a multistate process, with ligand binding increasing apparent folding stability, primarily due to aggregation.
Conclusions:
- Folate binding significantly alters FBP secondary structure and induces fluorescence quenching.
- FBP exhibits a stable folding intermediate, and its unfolding pathway is complex.
- The observed increase in folding stability upon ligand binding is attributed to ligand-induced aggregation rather than enhanced intrinsic stability.