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 n o other phospholipid class. To probe mechanisms for membrane interactions a nd phosphatidylcholine binding, we expressed recombinant human PC-TP in Esc herichia coli using a synthetic gene. Optimization of codon usage for bacte rial protein translation increased expression of PC-TP from trace levels to >10% of the E. coli cytosolic protein mass. On the basis of secondary stru cture predictions of an amphipathic alpha -helix (residues 198-212) in prox imity to a hydrophobic alpha -helix (residues 184-193), we explored whether the C-terminus might interact with membranes and promote binding of phosph atidylcholines. Consistent with this possibility, truncation of five residu es 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 containin g one (196-214PC-TP) Or both (183-214PC-TP) predicted C-terminal alpha -hel ices in aqueous buffer were most consistent with random coil structures. Ho wever, both peptides adopted alpha -helical configurations in the presence of trifluoroethanol or phosphatidylcholine/phosphatidylserine small unilame llar vesicles. The helical content of 196-214PC-TP increased in proportion to vesicle phosphatidylserine content, consistent with stablization of the alpha -helix at the membrane surface. In contrast, the helical content of 1 83-214PC-TP was not influenced by Vesicle composition, implying that the mo re hydrophobic of the alpha -helices penetrated into the membrane bilayer. These studies suggest that tandem alpha -helices located near the C-terminu s of PC-TP facilitate membrane binding and extraction of phosphatidylcholin es.