In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >~150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.

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Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2) on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores, but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behaviour is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore, but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.

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A potentiometric titration study in aqueous solution (I = 0.10 mol dm-3, KCI) of the complexation of benzoic, 4-methylbenzoic and (RS)-2-phenylpropanoic acids (HA) and their conjugate bases (A-) with Bcyclodextrin, BCD, and its substituted analogue, 6A-amino-6A-deoxy-~-cyclodextrin, BCDNH, , in which a primary hydroxy group is replaced by an amino group which may be protonated to produce a singly charged species, BCDNH; , is reported. At 298.2 K the stability constants for the complexes have the values (in dm3 mol-') shown in parentheses : benzoic acid * BCD (K, HA = 590 & 60) ; benzoate PCD (K, A = 60 f 10) ; benzoic acid * BCDNH; (K,,, = 340 f 30) ; benzoate . BCDNH; ( K ~A = 120 & 20) ; benzoate . BCDNH, (K3A = 50 f 20) ; 4-methylbenzoic acid -PCD (K, HA = 1680 & 90) ; Cmethylbenzoate -BCD (K,, = 110 f 1) ; 4-methylbenzoic acid * BCDNH; ( K 2 H A = 910 f 20); 4-methylbenzoate -BCDNH; ( K 2 A = 330 & 20); and 4-methylbenzoate . PCDNH, (K3A = 100 & 20).These data indicate that for a given cyclodextrin the guest carboxylic acids form complexes of higher stability than do their conjugate base analogues, and that BCDNH; forms more stable complexes with the conjugate bases than do BCD and PCDNH,. These trends are also observed for the complexation of (RS)-2-phenylpropanoic acid and (RS)-2-phenylpropanoate where the complexes indicated are characterised by the stability constants (in dm3 mol-') shown in parentheses: (RS)-2-phenylpropanoic acid. BCD ( K , R H A = 1090 k 30, K,,,, = 1010 f 40) ; (RS)-2-phenylpropanoate * PCD (K, , , = 63 f 8, K,,, = 52 & 5) ; (RS)-2-phenylpropanoic K,,, = 110 f 10); and (RS)-2-phenylpropanoate BCDNH, (K3,, = 36 f 6, K3,A = 13 & 7). These data also show that while K1,HA and K i s H A , and K , , , and KISA are indistinguishable for (RS)-2-phenylpropanoic acid * BCD and (RS)-2-phenylpropanoate * BCD, chiral discrimination is indicated by K,,HA > K2sHA for (RS)-2-phenylpropanoic acid BCDNH;, K,,, > KZSA for (RS)-2-phenylpropanoate -PCDNH; , and K3,, > KBSA for (RS)-2phenylpropanoate -BCDNH, . The ' H NMR spectra of the methyl groups of the enantiomers of (RS)-2-phenylpropanoic acid appear as two separate doublets, indicating chiral discrimination when complexed by BCD or BCDNH; , but such chiral discrimination is not observed for (RS)-2-phenylpropanoate when complexed by PCDNH; . The implications of these observations are discussed. acid * BCDNH; (K2RHA = 580 f 20, K~,HA = 480 & 10); (RS)-2-phenylpropanoate * BCDNH;