TMH insertion in PFO and PFO<sup>R468A</sup>.

2013-02-20T02:57:27Z (GMT) by Kelley J. Dowd Rodney K. Tweten
<p>A cysteine was substituted for Ala-215 in PFO and PFO<sup>R468A</sup>, 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002787#ppat.1002787-Shepard1" target="_blank">[27]</a>. 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<sup>R468A</sup> were mixed in a 4∶1 molar ratio with PFO<sup>A215C-NBD</sup> and PFO<sup>R468A•A215C-NBD</sup> 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.</p>