High-level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain.
Phosphatidylcholine transfer protein (PC-TP) is a 214-amino acid cytosolic protein that promotes intermembrane transfer of phosphatidylcholines, but no other phospholipid class. To probe mechanisms for membrane interactions and phosphatidylcholine binding, we expressed recombinant human PC-TP in Escherichia coli using a synthetic gene. Optimization of codon usage for bacterial protein translation increased expression of PC-TP from trace levels to >10% of the E. coli cytosolic protein mass. On the basis of secondary structure predictions of an amphipathic alpha-helix (residues 198-212) in proximity to a hydrophobic alpha-helix (residues 184-193), we explored whether the C-terminus might interact with membranes and promote binding of phosphatidylcholines. Consistent with this possibility, truncation of five residues from the C-terminus shortened the predicted amphipathic alpha-helix and decreased PC-TP activity by 50%, whereas removal of 10 residues eliminated the alpha-helix, abolished activity, and markedly decreased the level of membrane binding. Circular dichroic spectra of synthetic peptides containing one ((196-214)PC-TP) or both ((183-214)PC-TP) predicted C-terminal alpha-helices in aqueous buffer were most consistent with random coil structures. However, both peptides adopted alpha-helical configurations in the presence of trifluoroethanol or phosphatidylcholine/phosphatidylserine small unilamellar vesicles. The helical content of (196-214)PC-TP increased in proportion to vesicle phosphatidylserine content, consistent with stabilization of the alpha-helix at the membrane surface. In contrast, the helical content of (183-214)PC-TP was not influenced by vesicle composition, implying that the more hydrophobic of the alpha-helices penetrated into the membrane bilayer. These studies suggest that tandem alpha-helices located near the C-terminus of PC-TP facilitate membrane binding and extraction of phosphatidylcholines.
Feng
L
L
Department of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Chan
W W
WW
Roderick
S L
SL
Cohen
D E
DE
D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D049488:Phosphatidylethanolamine Binding Protein; D049294:Phospholipid Transfer Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000727:Androgen-Binding Protein; D002352:Carrier Proteins; C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D049294:Phospholipid Transfer Proteins; D011994:Recombinant Proteins