Interaction of a nonspecific wheat lipid transfer protein with phospholipid monolayers imaged by fluorescence microscopy and studied by infrared spectroscopy

Abstract

The interaction of a nonspecific wheat lipid transfer protein (LTP) with phospholipids has been studied using the monolayer technique as a simplified model of biological membranes. The molecular organization of the LTP-phospholipid monolayer has been determined by using polarized attenuated total internal reflectance infrared spectroscopy, and detailed information on the microstructure of the mixed films has been investigated by using epifluorescence microscopy. The results show that the incorporation of wheat LTP within the lipid monolayers is surface-pressure dependent. When LTP is injected into the subphase under a dipalmytoylphosphatidylglycerol monolayer at low surface pressure (< 20 mN/m), insertion of the protein within the lipid monolayer leads to an expansion of dipalmytoylphosphatidylglycerol surface area. This incorporation leads to a decrease in the conformational order of the lipid acyl chains and results in an increase in the size of the solid lipid domains, suggesting that LTP penetrates both expanded and solid domains. By contrast, when the protein is injected under the lipid at high surface pressure (> or = 20 mN/m) the presence of LTP leads neither to an increase of molecular area nor to a change of the lipid order, even though some protein molecules are bound to the surface of the monolayer, which leads to an increase of the exposure of the lipid ester groups to the aqueous environment. On the other hand, the conformation of LTP, as well as the orientation of alpha-helices, is surface-pressure dependent. At low surface pressure, the alpha-helices inserted into the monolayers are rather parallel to the monolayer plane. In contrast, at high surface pressure, the alpha-helices bound to the surface of the monolayers are neither parallel nor perpendicular to the interface but in an oblique orientation.

Homology modelling and molecular dynamics aided analysis of ligand complexes demonstrates functional properties of lipid‐transfer proteins encoded by the barley low‐temperature‐inducible gene family, blt4

“…In the maize LTP, Lys46 was proposed to bind the phosphate group of lysophosphatidyl choline, while other basic residues are predicted to have a crucial function in the adsorption of LTPs on negatively charged lipid membrane surfaces. This prediction is supported by the finding that lipid transfer activity is improved when the anionic phosphatidyl glycerol content of the membrane is increased (Petit et al, 1994) and the demonstration by infrared spectroscopy of interaction between lysine δ-amino groups of maize LTP and ester groups of di-palmitoyl phosphatidylglycerol monolayers (Subirade et al, 1995). Figure 2 (lower model) shows the amino acid sequence differences (coloured green) between BLT4.9 and the maize LTPs, which affect surface charge properties (10 positively charged residues are detected on BLT4.9 while only eight of them exist on the maize LTP).…”

“…The small hydrophobic residues Ala37 and Gly38 of BLT4.9 are replaced by the bulkier and polar Asn37 and Asn38 in the maize protein, which results in a narrower entrance hole. In addition, Ala37 and Gly38 together with Val27 in BLT4.9 (Ala27 in maize) create a hydrophobic surface patch which may be involved in lipid binding and/or interactions with membranes (Subirade et al, 1995). The two extra positively charged residues (Arg35 and Arg90) in all three BLT4 proteins corresponding to the non-charged Ser35 and Asn93 of the maize LTP, are also located in close vicinity to the binding tunnel.…”

Infrared studies of protein-induced perturbation of lipids in lipoproteins and membranes

“…However, infrared spectroscopy can also be applied to the study of lipids spread as monolayers at an airwater interface. Subirade et al (1995) combined IR spectroscopy and fluorescence microscopy in a study of the interaction of a nonspecific wheat lipid transfer protein with phosphatidylglycerol monolayers. This is another example of a basic soluble protein that interacts with phospholipids.…”

Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds

“…Until now, it was impossible to isolate a stable complex involving diacyl phospholipids and to establish the mode of binding of such a molecule to the ns-LTP. Recent monolayer experiments (Subirade et al, 1995) give some new information on the interaction between the wheat ns-LTP and diacylphospholipids. A model is proposed involving a collisional complex-shuttle mechanism.…”