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-β-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 α-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an α-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) α-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.
Skip Nav Destination
Article navigation
Research Article|
May 15 2004
Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast
Pierre DOUETTE;
Pierre DOUETTE
*Laboratory of Bioenergetics, Centre for Oxygen Research and Development, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium
Search for other works by this author on:
Rachel NAVET;
Rachel NAVET
*Laboratory of Bioenergetics, Centre for Oxygen Research and Development, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium
Search for other works by this author on:
Fabrice BOUILLENNE;
Fabrice BOUILLENNE
†Centre for Protein Engineering, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium
Search for other works by this author on:
Alain BRANS;
Alain BRANS
†Centre for Protein Engineering, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium
Search for other works by this author on:
Claudine SLUSE-GOFFART;
Claudine SLUSE-GOFFART
*Laboratory of Bioenergetics, Centre for Oxygen Research and Development, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium
Search for other works by this author on:
André MATAGNE;
André MATAGNE
†Centre for Protein Engineering, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium
Search for other works by this author on:
Francis E. SLUSE
Francis E. SLUSE
1
*Laboratory of Bioenergetics, Centre for Oxygen Research and Development, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium
1To whom correspondence should be addressed (e-mail F.Sluse@ulg.ac.be).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
December 19 2003
Revision Received:
February 05 2004
Accepted:
February 06 2004
Accepted Manuscript online:
February 06 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 380 (1): 139–145.
Article history
Received:
December 19 2003
Revision Received:
February 05 2004
Accepted:
February 06 2004
Accepted Manuscript online:
February 06 2004
Citation
Pierre DOUETTE, Rachel NAVET, Fabrice BOUILLENNE, Alain BRANS, Claudine SLUSE-GOFFART, André MATAGNE, Francis E. SLUSE; Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast. Biochem J 15 May 2004; 380 (1): 139–145. doi: https://doi.org/10.1042/bj20031957
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.