Reference : Secondary-structure characterization by far-UV CD of highly purified uncoupling protein ...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/3714
Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast
English
Douette, Pierre [Université de Liège - ULg > > Bioénergétique et physiologie cellulaire >]
Navet, Rachel mailto [Université de Liège - ULg > > Bioénergétique et physiologie cellulaire > >]
Bouillenne, Fabrice mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines >]
Brans, Alain mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines >]
Sluse-Goffart, C. [Université de Liège - ULg > > Bioénergétique et physiologie cellulaire > >]
Matagne, André mailto [Université de Liège - ULg > Département des sciences de la vie > Enzymologie >]
Sluse, Francis mailto [Université de Liège - ULg > Département des sciences de la vie > Bioénergétique et physiologie cellulaire >]
May-2004
Biochemical Journal
Portland Press
380
Pt 1
139-145
Yes (verified by ORBi)
International
0264-6021
1470-8728
London
United Kingdom
[en] CD ; fluorescence resonance energy transfer ; homology modelling ; mitochondrial membrane protein ; secondary structure ; uncoupling protein
[en] The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His(6) epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95 %). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCPI-His(6) retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of a-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68 %, which is at least 25 % higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.
http://hdl.handle.net/2268/3714
also: http://hdl.handle.net/2268/33058
10.1042/BJ20031957

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