Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to β-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12–β-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the β-oxidation pathway, a binding 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. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty 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 tetracosanoic acid (24:0) was negligible.
High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2)
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Tobias B. DANSEN, Jan WESTERMAN, Fred S. WOUTERS, Ronald J. A. WANDERS, Arie van HOEK, Theodorus W. J. GADELLA, Karel W. A. WIRTZ; High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2). Biochem J 1 April 1999; 339 (1): 193–199. doi: https://doi.org/10.1042/bj3390193
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