The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding.
Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.
de Brouwer
A P
AP
Department of Biochemistry of Lipids, Institute of Biomembranes, Padualaan 8, 3584 CH Utrecht, The Netherlands. a.p.m.debrouwer@chem.uu.nl
Bouma
B
B
van Tiel
C M
CM
Heerma
W
W
Brouwers
J F
JF
Bevers
L E
LE
Westerman
J
J
Roelofsen
B
B
Wirtz
K W
KW
D000727:Androgen-Binding Protein; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D019281:Dimerization; D004926:Escherichia coli; D006639:Histidine / Q000096:biosynthesis; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000737:chemistry; D008239:Lysine / Q000502:physiology; D010713:Phosphatidylcholines / Q000096:biosynthesis / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D000727:Androgen-Binding Protein; D002352:Carrier Proteins; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D006639:Histidine; D008239:Lysine