CD; fluorescence resonance energy transfer; homology modelling; mitochondrial membrane protein; secondary structure; uncoupling protein
Abstract :
[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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Douette, Pierre ; Université de Liège - ULiège > Bioénergétique et physiologie cellulaire
Navet, Rachel ; Université de Liège - ULiège > Bioénergétique et physiologie cellulaire
Bouillenne, Fabrice ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Brans, Alain ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Sluse-Goffart, Claudine; Université de Liège - ULiège > Bioénergétique et physiologie cellulaire
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie
Sluse, Francis ; Université de Liège - ULiège > Département des sciences de la vie > Bioénergétique et physiologie cellulaire
Language :
English
Title :
Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast
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