High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2)

Binding of fluorescent fatty acids to bovine liver non-specific lipid-trans fer protein (nsL-TP) was assessed by measuring fluorescence resonance energ y transfer (FRET) between the single tryptophan residue of nsL-TP and the f luorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parin aric acid to nsL-TP, FRET was observed indicating that these fatty acids we re accommodated in the lipid binding site closely positioned to the tryptop han residue. Substantial binding was observed only when these fatty acids w ere presented in the monomeric form complexed to beta-cyclodextrin. As show n by lime-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the d irect molecular environment of the pyrene moiety: i.e. the fluorescence lif etime of the directly excited pyrene increased at least by 25% and a distin ct rotational correlation time of 7 ns was observed. In order to evaluate t he affinity of nsL-TP for intermediates of the beta-oxidation pathway, a bi nding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanopl-CoA and 2-hexadecenoyl-CoA were found to bind readil y to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA b ound poorly. The highest affinities were observed for the very-long-chain f atty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1 -CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosan oic acid (24:0) was negligible.