TMH insertion in PFO and PFO^R468A.

A cysteine was substituted for Ala-215 in PFO and PFO^R468A, which is located in TMH1.The sidechain of Ala-215 is in an aqueous environment in the soluble monomer, but enters the membrane upon formation of the membrane spanning β-barrel [27]. Therefore an NBD probe positioned at this site undergoes a polar to nonpolar transition that is detected by an increase in the fluorescence emission of the probe.(A) Each NBD-labeled derivative was incubated in the presence (dashed line) and absence (solid line) of human erythrocyte ghost membranes. (B) Unlabeled native PFO or PFO^R468A were mixed in a 4∶1 molar ratio with PFO^A215C-NBD and PFO^{R468A•A215C-NBD} derivatives. The fluorescence emission for all experiments wasrelative to the maximum emission change observed for NBD-labeled PFO. The fluorescence emission intensity of NBD was measured from 500–600 nm. The data are representative of 3 experiments.