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Lee, Yang, Willers, Chrissie, Kunji, Edmund R S and Crichton, Paul G. 
ORCID: https://orcid.org/0000-0003-3786-8359
(2015)
Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin.
Proceedings of the National Academy of Sciences of the United States of America (PNAS), 112 (22).
pp. 6973-6978.
ISSN 0027-8424
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Abstract
Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria.
| Item Type: | Article |
|---|---|
| Additional Information: | Freely available online through the PNAS open access option. |
| Uncontrolled Keywords: | brown fat,cardiolipin binding,membrane protein,nucleotide binding,oligomeric state,sdg 3 - good health and well-being ,/dk/atira/pure/sustainabledevelopmentgoals/good_health_and_well_being |
| Faculty \ School: | Faculty of Medicine and Health Sciences > Norwich Medical School |
| UEA Research Groups: | Faculty of Medicine and Health Sciences > Research Groups > Cardiovascular and Metabolic Health Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health |
| Related URLs: | |
| Depositing User: | Pure Connector |
| Date Deposited: | 03 Nov 2016 12:00 |
| Last Modified: | 19 Oct 2023 01:51 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/61224 |
| DOI: | 10.1073/pnas.1503833112 |
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