Fatty acid binding site of mitochondrial uncoupling protein UCP2 as probed by EPR spectroscopy of spin-labeled fatty acids

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled fatty acids was used to investigate their interaction withEscherichia coli-expressed human mitochondrial uncoupling protein UCP2 refolded from inclusion bodies in nonaethylene glycol monododecyl ether (C12E9) micelles. 5-DOXYL-steari...

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Bibliographic Details
Published in:Applied magnetic resonance 2006-06, Vol.30 (3-4), p.373-383
Main Authors: Raju, N., Špaček, T., Ježek, J., Caminiti, I. M., Leinisch, F., Hideg, K., Ježek, P., Trommer, W. E.
Format: Article
Language:English
Subjects:
Arachidonic acid
Binding sites
Electron paramagnetic resonance
Electron spin
Fatty acids
Micelles
Palmitic acid
Proteins
Spectroscopy
Spectrum analysis
Stearic acid
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Summary:Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled fatty acids was used to investigate their interaction withEscherichia coli-expressed human mitochondrial uncoupling protein UCP2 refolded from inclusion bodies in nonaethylene glycol monododecyl ether (C12E9) micelles. 5-DOXYL-stearic acid and 4-PROXYL-palmitic acid bound to UCP2 exhibited additional clearly separated h+1I, h−1I “immobile” peaks in the low- and high-field region, respectively, separated by 42 and 44 Gauss, and extensively reduced h+1M, h−1M “mobile” peaks, separated by about 30 G, whereas with 7-DOXYL-stearic acid the I and M peaks were smoothed together into one wide peak. Competition of 4-PROXYL-palmitic acid with added palmitic acid, arachidonic acid, and all-cis-8,11,14-eicosatrienoic acid and of 7-DOXYL-stearic acid with arachidonic acid was indicated by the disappearance of the h+1I, h−1I “immobile” peaks, whereas redistribution in micelles without protein was indicated by the rising of the h+1M, h−1M “mobile” peaks. In conclusion, a competition of palmitic, arachidonic, and eicosatrienoic acid within a putative fatty acid binding site was observed for mitochondrial uncoupling protein UCP2. This finding together with the observation of EPR spectra of highly immobilized probes exclusively in the presence of the recombinant UCP2 suggest the existence of a fatty acid binding site on UCP2 which is a prerequisite of the fatty acid cycling mechanism as previously postulated for UCP1.
ISSN:0937-9347
1613-7507
DOI:10.1007/BF03166207