Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast.
The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 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 UCP1-His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-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.
Douette
Pierre
P
Laboratory of Bioenergetics, Centre for Oxygen Research and Development, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium.
Navet
Rachel
R
Bouillenne
Fabrice
F
Brans
Alain
A
Sluse-Goffart
Claudine
C
Matagne
André
A
Sluse
Francis E
FE
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D007473:Ion Channels; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008928:Mitochondria / Q000378:metabolism; D008929:Mitochondria, Heart / Q000737:chemistry; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; C000604373:Ucp1 protein, rat; D000071256:Uncoupling Protein 1; D000226:Mitochondrial ADP, ATP Translocases