Fluorescent, Synthetic Amphiphilic Heptapeptide Anion Transporters: Evidence for Self‐Assembly and Membrane Localization in Liposomes

Abstract

Synthetic anion transporters (SATs) of the general type (n-C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-O-n-C7H15, 1, are amphiphilic peptides that form anion-conducting pores in bilayer membranes. To better understand membrane insertion, assembly and aggregation dynamics, and membrane penetration, four novel fluorescent structures were prepared for use in both aqueous buffer and phospholipid bilayers. The fluorescent residues pyrene, indole, dansyl, and NBD were incorporated into 1 to give 2, 3, 4, and 5, respectively. Assembly of peptide amphiphiles in buffer was confirmed by monitoring changes in the pyrene monomer/excimer peaks observed for 2. Solvent-dependent fluorescence changes that were observed for indole (3) and dansyl (4) side-chained SATs in bilayers showed that these residues experienced an environment between epsilon=9 (CH2Cl2) and epsilon=24 (EtOH) in polarity. Fluorescence resonance energy transfer (FRET) between 2 and 3 demonstrated aggregation of SAT monomers within the bilayer. This self-assembly led to pore formation, which was detected as Cl(-) release from the liposomes. The results of acrylamide quenching of fluorescent SATs supported membrane insertion. Studies with NBD-labeled SAT 5 showed that peptide partition into the bilayer is relatively slow. Dithionite quenching of NBD-SATs suggests that the amphiphilic peptides are primarily in the bilayer's outer leaflet. Images obtained by using a fluorescence microscope revealed membrane localization of a fluorescent SAT. Taken together, this study helps define the insertion, membrane localization, and aggregation behavior of this family of synthetic anion transporters in liposomal bilayers.

Design, Synthesis and Characterisation of a Fluorescently Labelled CyPLOS Ionophore

“…Similar linear correlations were also obtained with different polarity scales, such as Z , or by plotting the λ max values against the dielectric constants ( ε ) of the solvents. However, the solvent polarity parameter E T is determined directly by spectral measurements in solution and so its use appears more appropriate in the present case 11a…”

Using small molecules to facilitate exchange of bicarbonate and chloride anions across liposomal membranes

“…7,8 Despite the obvious importance of transmembrane bicarbonate transport, there have been no reports, to the best of our knowledge, of "small" molecules that are capable of promoting bicarbonate transport across lipid membranes, in contradistinction to the growing body of work on chloride and HCl transport across phospholipid vesicle membranes. [9][10][11][12][13][14][15] Phospholipid vesicles have been extensively investigated because of their usefulness as models for biological membranes. Unilamellar vesicles of a specific size are easily produced with control of the entrapped solution.…”

Aggregation and Supramolecular Membrane Interactions that Influence Anion Transport in Tryptophan‐Containing Synthetic Peptides

“…Using this difference in behavior, we determined that about 35 % of an NBD-substituted SAT partitioned into the bilayer. [15] Formation of aggregated pores was confirmed by fluorescence resonance energy transfer from a tryptophan-substituted SAT to a pyrenyl-SAT.…”

“…In the work reported here, we have incorporated tryptophan [15] in the heptapeptide sequence. Tryptophan is the rarest of the 20 common amino acids, [16] but occurs in proteins such as the KcsA potassium channel exclusively near the membrane-aqueous interfaces.…”