Binding of fatty acids to the uncoupling protein from brown adipose tissue mitochondria

The uncoupling protein 1 (UCP1) is a H+ carrier which plays a key role in heat generation in brown adipose tissue. The H+ transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2′-O-dansyl guanosine 5′-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2′-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants Ki were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10–14) and unsaturated C18 fatty acids exhibit stronger affinity (Ki=65 μM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.