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- Materials and methods
The binding of two mono-acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C-terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1-myristoylglycerophosphatidylcholine (MyrGro-PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 µm and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro-PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen-addad, C. & Pebay-Peyroula, E. (1999) Eur. J. Biochem.264, 562–568.]. Results from isothermal titration calorimetry experiments indicated non-classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two ω-hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.
Plant non-specific lipid transfer proteins (ns-LTPs) are well known for their ability to bind and transfer lipids [1,2]. They exhibit a basic pI and a 9-kDa molecular mass ; eight cysteines all involved in disulphide bridges help in maintaining the structure of the protein. The three-dimensional structure of ns-LTP1 has been determined by 1H-NMR and crystallography and reveals a hydrophobic cavity within the protein [4–9]. Lersche et al.  were the first to report a binding constant, Kd, in the case of barley LTP1 with fatty acids, lysophosphatidylcholine and acyl-CoA. In contrast with wheat LTP1 , the affinity was very low with binding constants in the range 10−2 to 10−4 m, except for acyl-CoA where Kd = 10−6 m. In that study, the intrinsic tyrosine fluorescence was used to probe the binding of lipids. However, it will be shown herein that this method is not well suited in the case of barley because its LTP1 possesses three tyrosines, one of which is solvent exposed and is not sensitive to lipid binding. Kader and colleagues have reported binding of lipids by maize LTP1 by using displacement fluorescence methods that involve labelled lipids [11,12]. The binding of two lipid monomers was suspected from these experiments. However, it was not possible to determine any binding constants from these data [11,12]. More recently, we analysed the binding constant of wheat LTP1 complexed with several lipids as obtained by intrinsic tyrosine fluorescence . Our data confirmed the lack of specificity for fatty acids and phospholipids with various chain lengths, with Kd values of about 10−6 m. With the finding that wheat LTP1 is capable of binding two lipids [9,11,13], it becomes a new goal to determine whether it is a general feature of the LTP1 family. For this purpose, barley LTP1 is well adapted because the free protein possesses one of the smallest cavity volumes, as shown by comparing several structures of LTP1 . Moreover, several authors have proposed that these proteins could be involved in the formation of polyesters [15–17]. Then, binding of two ω-hydroxylated lipids, an abundant cutin monomer, represents an interesting task and should help in our understanding of the physiological role of this protein family.
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- Materials and methods
In the case of wheat LTP1, intrinsic tyrosine fluorescence is well suited for monitoring the binding of ligands. This protein possesses two tyrosine residues and only one, Tyr79, is suspected to be involved in the signal enhancement upon titration . Barley LTP1 has an additional tyrosine residue in its sequence at the C-terminal position, Tyr91. As revealed by the three-dimensional structure , Tyr91 is solvent exposed so that its fluorescence emission is not partially quenched as is the case for the other tyrosine residues (Tyr16 and Tyr79), which are embedded within the core of the protein. As a consequence, Tyr91 mainly contributes to the total fluorescence emission of barley LTP1, which is markedly higher than that of wheat LTP1 (Fig. 1) which lacks this terminal tyrosine residue. Moreover, Tyr91 hides the Tyr79 enhancement of fluorescence that is generated on lipid binding so that titration cannot be performed under these conditions. This could explain the reason for the poor affinity reported in the case of complex formation of barley LTP1 with various lipids . However, the removal of Tyr91 can be easily achieved by using a carboxypeptidase A even if it also hydrolyses Ile90. It should be noted that the structure of this truncated protein is not too affected by this procedure. Indeed, despite the carboxyl terminal has been shown to interact with Arg44 and contributes to the stability of the structure , this salt bridge does not always occur (see 1BE2.pdb ). In contrast with the native barley LTP1, this truncated protein can be used for monitoring the binding of lipids. Titration gives rise to an increase of fluorescence as obtained in the case of wheat LTP1. Our result shows an affinity comparable with that obtained for the complex formation between wheat LTP1 and various lipids, indicating that the interaction seems to be rather non-specific as long as the binding ligand possesses a hydrophobic tail. These similarities are consistent with the results of structural studies that have shown that the presence of the lipid polar head does not contribute significantly to the complex stability [7,9].
Of considerable interest is the finding that barley LTP1 is also capable of binding two lipids. This result is well illustrated by molecular modelling, which shows that both lipids can fit within the cavity. It is clearly shown that the loading of two monomers is almost only associated with a shift of the C-terminus. Moreover, it is known that this region is flexible as revealed by temperature factors from crystallography .
The fact that the value returned by fitting the fluorescence data (n = 1.7) remains lower than two indicates that both lipids do not contribute in the same way to the increase of fluorescence. In another way, as in the case of wheat LTP1, it is probable that both sites exhibit close but not identical affinity . The binding of two monomers appears rather surprising if considering the structure of the complex with palmitic acid or palmitoyl-CoA [7,26]. In that case, it was clearly shown that only one monomer of lipid binds within the cavity. Such a result was also reported in the case of maize with one palmitic acid  or wheat with one MyrGro-PCho (F. Vovelle, CBM, Orleans, France, personal communication), while it is only recently that a structure of LTP1 complexed with two lipids has been published . The reason why some studies have shown that LTP1 binds one monomer while others indicate that two can be bound remains obscure. It is tempting to speculate that binding of two monomers could strongly depend on the experimental conditions such as pH, buffers, temperature or lipid and protein concentrations.
As perturbation of the tyrosine fluorescence can be used to monitor the binding of ligands to LTP1, ITC also represents a promising technique for such studies. The binding experiment of LTP1 with MyrGro-PCho reveals non-classic behaviour with an endothermic event at the end of the titration. This apparently results from the superimposition of exothermic and endothermic behaviours. However, this cannot be fitted with the binding model so information is unavailable about the individual sites.
In the hypothesis of the participation of LTP1 in the biosynthesis of cutin [15,17], a binding study involving ω-hydroxypalmitic acid represents an attractive investigation. The affinity of this lipid for the barley LTP1 is analogous with that reported in the case of wheat . Moreover, in the same way, n is closer to two compared with values obtained with other lipids. This suggests that in this case, both sites exhibit similar affinity. This result is of strong importance if considering the head-to-tail orientation of both monomers within the protein cavity. An analogy with the structure derived from that of wheat suggests that the ω-hydroxyl group of monomer B would be located close to the carboxyl group of monomer A.
We have clearly shown that barley LTP1, which possesses a small hydrophobic cavity in its free state, is capable of binding two monomers of MyrGro-PCho. This behaviour probably depends on the biochemical conditions. This result, together with the binding of a hydroxylated fatty acid, strongly emphasizes the participation of LTP1 in cutin biosynthesis.