Steady-state tyrosine fluorescence to study the lipid-binding properties of a wheat non-specific lipid-transfer protein (nsLTP1)

The binding properties of a wheat non-specific lipid-transfer protein (nsLT P1) for different mono- and diacylated lipids was investigated. Lipids vari ed by their chain length, unsaturation and/or polar head group. In the case of fatty acid or lysophospholipid with a C10 chain length, no interaction can be measured, while poor affinity is reported for a C12 chain length. Th e dissociation constant (K-d) is about 0.5 mu M independent of chain length from C14 to C18. The same affinity is obtained for Cls fatty acids with on e or two unsaturations, whatever the cis-trans double bond isomery. In all cases, the number of binding sites, n, by protein ranges between 1.6 and 1. 9, suggesting that two lipids can fit within the protein. omega-Hydroxy-pal mitic acid, a natural monomer of cutin polymer, is found to interact with n sLTP1 with a K-d Of 1 mu M and n = 2. In contrast with previous data that r eported the binding of the anionic diacylated phospholipid, DMPG (Sodano et al., FEES Lett. 416 (1997) 130-134), nsLTP1 is not able to bind dimyristoy lphosphatidylcholine, dimyristoylphosphatidic acid, palmitoyl-oleoylphospha tidylcholine or palmitoyl-oleoylphosphatidylglycerol added as liposomes or solubilized in ethanol. However, when both nsLTP1 and lipids are first solu bilized in methanol, and then in the buffer, it was evidenced that the prot ein can bind these lipids. These results suggest that lipid-lipid interacti ons play an essential role in the binding process of plant nsLTP1 as previo usly mentioned for other lipid-transfer proteins. (C) 2000 Elsevier Science B.V. All rights reserved.