Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.
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 microM 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 omega-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.
Douliez
J P
JP
Laboratoire de Biochimie et Technologie des Protéines, INRA, rue de la Géraudière, BP 1627, 44316 Nantes, France. douliez@nantes.inra.fr
Jégou
S
S
Pato
C
C
Mollé
D
D
Tran
V
V
Marion
D
D
D002151:Calorimetry / Q000379:methods; D002352:Carrier Proteins / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins; D001467:Hordeum; D008024:Ligands; D008244:Lysophosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010169:Palmitic Acids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D017186:Titrimetry / Q000379:methods; D014443:Tyrosine / Q000737:chemistry
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D008024:Ligands; C422456:Ltp1 protein, barley; D008244:Lysophosphatidylcholines; D010169:Palmitic Acids; C035255:myristoyllysophosphatidylcholine; D014443:Tyrosine; C063407:16-hydroxypalmitic acid