Identification of the Lipid-Binding Site of Phosphatidylcholine-Transfer Protein with Phosphatidylcholine Analogs Containing Photactivable Carbene Precursors

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Abstract
The lipid binding site of the phosphatidylcholine transfer protein from bovine liver has been investigated by use of phosphatidylcholine analogs which carry a diazirinophenoxy group linked to the ω-carbon of either the sn-2-[1-14C]hexanoyl (PCI) or sn-2-[1-14C]undecanoyl chain (PC II). Photolysis of the PCI(PCII)-transfer protein complex resulted in a covalent coupling of 30–40% of the label to the protein as shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Upon mild alkaline treatment of the photolysed complex the protein containing covalently coupled 14C-label was separated fro the noncoupled 14C-label by gel permeation chromatography. The 14C-labeled protein was degraded with protease from Staphylococcus aureus, trypsin and cyanogen bromide and specific 14C-labeled peptides were sequenced by automated Edman degradation. Major sites of coupling shown by release of radioactivity were identified as Tyr54 and the peptide segment Val171-Phe-Met-Tyr-Tyr-Phe-Asp177. Both PC I and PC II coupled extensively to Tyr54 (90% and 5% of total labeling, respectively). The remainder of the radioactivity was released from the peptide Val171-Asp177 with a distinct difference in in the pattern of release depending on whether PC I or PC II were used. Thus, coupling occurred preferentially to Tyr175 and Asp177 with PC I while Val171 and Met173 were labeled preferentially with PC II. This shift in coupling is compatible with an increasae of 0.6 nm for the sn-2-fatty-acyk cgaubs if OC I and II, assuming that the peptide Val171-Asp177 has adopted the strongly predicted β-strand configuration. These data have beeninterpreted in terms of the localization of phosphatidylcholine in the phosphatidylcholine transfer protein.