Inner Field Compensation as a Tool for the Characterization of Asymmetric Membranes and Peptide-Membrane Interactions  Sven O. Hagge, Andre Wiese, Ulrich Seydel, Thomas Gutsmann  Biophysical Journal  Volume 86, Issue 2, Pages 913-922 (February 2004) DOI: 10.1016/S0006-3495(04)74167-3 Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 1 Potential profile across an asymmetric bilayer membrane (adopted from Schoch et al. (1979)). Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 2 Chemical structure of deep rough mutant LPS from E. coli strain F515, S. Minnesota strain R595, and P. mirabilis strain R45. Negative charges are indicated by a minus within a black circle, positive charges by a plus within a white circle. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 3 Voltage-dependence of the capacitance, amplitude, and phase of the second harmonic (A). For inner field compensation measurements, an ac excitation is applied to planar lipid membranes resulting in a current response in the first and the second harmonic (I1≠0, I2≠0) (B). When applying an external transmembrane voltage U=−ΔΨ, the second harmonic vanishes (I1≠0, I2=0) (C) and the inner membrane potential difference ΔΨ′ resulting from the inner membrane and the transmembrane potential profile is zero. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 4 Comparison of the inner membrane potential differences ΔΦ (light gray bars) determined by carrier transport measurements and ΔΨ by IFC measurements (dark gray bars), respectively, for asymmetric membrane, composed of different phospholipids or lipopolysaccharides. In the figure only one leaflet is defined, the other one was from PL in all cases. The PL side consists of 81mol % PE, 17mol % PG, and 2mol % DPG. Measurements were performed at 37°C in electrolyte solution containing 100mM KCl and 5mM MgCl2 buffered with 5mM HEPES and adjusted to pH 7. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 5 Change of the capacitance (gray traces) and of the inner membrane potential difference ΔΨ (black traces) of different membranes after addition of rCAP18106–137. Peptide was added 5min after membrane preparation (arrow): LPS F515/PL (A), LPS R45/PL (B), and DPhyPC/PL (C). Composition of PL side and electrolyte solution as described for Fig. 4. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 6 Change of the capacitance (gray traces) and the inner membrane potential difference ΔΨ (black traces) of F515 LPS/PL membranes after addition of rCAP1898–117 (A) and rCAP18106–125 (B). Peptide was added 5min after membrane preparation (arrow). Composition of PL side and electrolyte solution as described for Fig. 4. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 7 Schematic membrane potential profile across asymmetric PS/PL (A) and LPS/PL (B) membranes on the basis of the Schoch model. The negative charges of the lipid molecules are indicated by black circles. The arrows indicate the polarity of ΔΦ and ΔΨ. The exact potential profile in the headgroups of the LPS molecules is unknown; therefore, we indicated the possible range by a solid line and a dotted line. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 8 Correlation between surface charge density σ of the side of peptide addition and change of the inner membrane potential difference ΔΨ of various LPS/PL and phospholipid PL membranes. Composition of PL side and electrolyte solution as described for Fig. 4. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 9 Correlation between charge of the peptides Q and change of the inner membrane potential difference ΔΨ of LPS F515/PL membranes. Composition of PL side and electrolyte solution as described for Fig. 4. Biophysical Journal 2004 86, 913-922DOI: (10.1016/S0006-3495(04)74167-3) Copyright © 2004 The Biophysical Society Terms and Conditions