Identification of the lipid-binding site of phosphatidylcholine-transfer protein with phosphatidylcholine analogs containing photoactivable carbene precursors.
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 omega-carbon of either the sn-2-[1-14C]hexanoyl (PC I) or sn-2-[1-14C]undecanoyl chain (PC II). Photolysis of the PC I(PC II)-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 from 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 50% 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 increase of 0.6 nm for the sn-2-fatty-acyl chains of PC I and II, assuming that the peptide Val171-Asp177 has adopted the strongly predicted beta-strand configuration. These data have been interpreted in terms of the localization of phosphatidylcholine in the phosphatidylcholine transfer protein.
D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D006838:Hydrocarbons; D050356:Lipid Metabolism; D008099:Liver / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives*; D010446:Peptide Fragments / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010777:Photochemistry; D013050:Spectrometry, Fluorescence
D000727:Androgen-Binding Protein; D002352:Carrier Proteins; D006838:Hydrocarbons; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; C030011:carbene; D008697:Methane