Characterisation of acyl binding by a plant lipid-transfer protein.
Maize lipid-transfer protein (LTP) is a small soluble protein which is able to transfer in vitro phospholipids between membranes and to bind fatty acids or lysoderivatives. In the studies reported here, fluorescent-labelled fatty acids were used to characterise the nature of the binding site on LTP. A fluorescent analogue of 12 carbons with a pyrene moiety attached at the end, alone or in conjunction with an anthroyloxy analogue, indicated that LTP could bind two fatty acids although with a marked difference in affinity. The binding capacity was strongly affected after reduction of the protein by dithiothreitol, showing that the four S-S bonds of LTP are essential for its lipid binding property. Other analogues used were 16-carbon or 18-carbon fatty acids with an anthracene moiety attached at different points of the hydrocarbon chain. Emission maxima of these molecules varied with the analogue and suggested a motional constraint for the bound fatty acid which is more important around the middle of the chain than at its extremities. Binding displacement studies were carried out with a wide range of fatty acids or fatty acyl derivatives. Fatty acids of 16 to 19 carbons were found to be the preferred ligands. The presence of one double bond did not change appreciably the affinity of LTP, although the presence of two or three double bonds or of a hydroxyl moiety significantly reduced the affinity. Fatty acyl-CoA or lysoderivatives bound as well as the corresponding fatty acid.
Zachowski
A
A
Laboratoire de Physiologie Cellulaire et Moléculaire, UMR CNRS 7632, Université Pierre et Marie Curie, Paris, France. Alain.Zachowski@snv.jussieu.fr
Guerbette
F
F
Grosbois
M
M
Jolliot-Croquin
A
A
Kader
J C
JC
D052179:Antigens, Plant; D002352:Carrier Proteins / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D003313:Zea mays / Q000378:metabolism*
D052179:Antigens, Plant; D002352:Carrier Proteins; D005227:Fatty Acids; D007850:Lauric Acids; D010940:Plant Proteins; C511920:lipid transfer proteins, plant; D004229:Dithiothreitol