![]() This is particularly surprising if they cross the bilayer as monomers, without formation of a transient pore that would allow the charges to remain hydrated as the peptide crosses the membrane. Intuitively, we tend to expect that cationic amphipathic peptides cannot move across a lipid bilayer spontaneously. Translocation efficiency drops for the motif peptides when P/L is decreased from 1:100 to 1:500, which is typical of membrane-active peptides but translocation increases for TP2 between P/L of 1:100 and 1:1000, which is unusual. Furthermore, experiments were performed at peptide-to-lipid ratios (P/L) as low as 1:500 for the motif peptides and 1:1000 for TP2, suggesting that monomers are the translocating species. Because these peptides cause little membrane perturbation, as evident from the lack of vesicle leakage, it is safe to conclude that pores are not involved in translocation. The authors found that the LRLLR motif translocates better than the parent TP2, but some of its arginine positional variants are even more efficient at crossing the lipid bilayer. TP2 was used as positive control and an observed negative (ONEG), the peptide PLGRPQLRRGQWC-amide was used as negative control for translocation. Further, Fuselier and Wimley tested the effect of the position of the two Arg residues within the LRLLR motif on peptide translocation. Release of Cys-NBD served as the assay for peptide translocation. This tag allows monitoring of the cleavage of the Trp-Cys bond by chymotrypsin encapsulated in large unilamellar vesicles of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In addition, a nitrobenzoxadiazole (NBD) fluorophore was attached to the thiol group of the C-terminal Cys (which is amidated). They incorporated the motif in the peptide LRLLRWC(NBD)-amide, which is fairly hydrophobic, but bears a +3 charge in a neutral solution. ![]() Here, Fuselier and Wimley ( 1) report the test of this hypothesis. They hypothesized that the LRLLR motif could be a minimal sequence that encodes the ability to translocate. Wimley and co-workers noticed that the central sequence in TP2, the motif LRLLR, was present in many of the translocating peptides selected by their screen. It was shown to cross the membranes of lipid vesicles and cells, even with polar fluorescent dyes attached ( 2, 5). TP2 is a 13-residue cationic peptide with the sequence PLIYLRLLRGQWC-amide. The translocating peptide TP2 emerged from this selection procedure as one of the most effective ( 2). This work follows a series of articles from Wimley’s laboratory on methods to reliably measure translocation of membrane-active peptides across phospholipid membranes, and on the use of an orthogonal high-throughput assay to screen combinatorial libraries of membrane-active peptides to specifically select for those peptides that spontaneously translocate but do not permeabilize membranes to water-soluble molecules ( 2, 3, 4). ![]() In this issue of the Biophysical Journal, Fuselier and Wimley ( 1) report the discovery of an amino acid sequence motif that appears to impart to polypeptides the ability to translocate across lipid membranes silently-that is, without causing flux of aqueous solutes across the membrane. E pur si muove (and yet it moves) is a comment attributed to Galileo after the Catholic Church forced him to recant his theory that the earth moved around the sun.
0 Comments
Leave a Reply. |