Orientation and Effects of Mastoparan X on Phospholipid Bicelles

Abstract

Mastoparan X (MPX: INWKGIAAMAKKLL-NH2) belongs to a family of ionophoric peptides found in wasp venom. Upon binding to the membrane, MPX increases the cell's permeability to cations leading to a disruption in the electrolyte balance and cell lysis. This process is thought to occur either through a membrane-thinning mechanism, where the peptide resides on the membrane surface thereby disrupting lipid packing, or through formation of an oligomeric pore. To address this issue, we have used both high-resolution and solid-state 2H NMR techniques to study the structure and orientation of MPX when associated with bicelles. NOESY and chemical shift analysis showed that in bicelles, MPX formed a well-structured amphipathic alpha-helix. In zwitterionic bicelles, the helical axis was found to rest generally perpendicular to the membrane normal, which could be consistent with the "carpet" mechanism for lytic activity. In anionic bicelles, on the other hand, the helical axis was generally parallel to the membrane normal, which is more consistent with the pore model for lytic activity. In addition, MPX caused significant disruption in lipid packing of the negatively charged phospholipids. Taken together, these results show that MPX associates differently with zwitterionic membranes, where it rests parallel to the surface, compared with negatively charged membranes, where it penetrates longitudinally.

Mechanism of Lipid Bilayer Disruption by the Human Antimicrobial Peptide, LL-37

“…LL-37-L was used in all the samples prepared for 13 C and 15 N NMR experiments. Samples for 31 P NMR, DSC, and CD experiments used LL-37-U. All of the peptide concentrations are reported as peptide/lipid mole ratio, or are given in mole% of peptide relative to the total moles of peptide plus lipid.…”

“…The raw data were converted to molar heat capacity using the CPCalc program provided with the calorimeter and the lipid concentration and molecular weight of each sample along with a partial specific volume of 0.988 mL/g (30). 31 P, 13 C, and 15 N NMR. 31 P, 13 C, and 15 N NMR spectra were acquired using a Varian/Chemagnetics 400 MHz spectrometer with 1 H, 31 P, 13 C, and 15 N frequencies of 400.139, 161.978, 100.618, and 40.551 MHz, respectively.…”

“…31 P, 13 C, and 15 N NMR. 31 P, 13 C, and 15 N NMR spectra were acquired using a Varian/Chemagnetics 400 MHz spectrometer with 1 H, 31 P, 13 C, and 15 N frequencies of 400.139, 161.978, 100.618, and 40.551 MHz, respectively. Home-built, double-resonance, flat-coil probes were used for the mechanically aligned samples with a coil just large enough to hold the samples.…”

“…Spectra were processed using Chemagnetics Spinsight software on a Sun Sparc workstation and Igor Pro 3.14 (Wavemetrics, Lake Oswego, OR) on an iMac. 31 P chemical shift spectra of mechanically aligned bilayer samples were obtained using a chemical shift echo sequence, 90°-τ-180°-τ-acq, with τ ) 100 µs and 25 kHz 1 H decoupling. Typical 31 P π/2 pulse lengths were 3-3.5 µs and the spectral width was 50 kHz.…”

“…In addition, the interaction of LL-37 with lipids containing choline headgroups is of interest because of the possible role of phosphorylcholine in bacterial resistance to LL-37 (24), although the presence of phosphatidylcholine headgroups on phospholipids does not prevent membrane disruption by LL-37. DMPC has also been widely used in other studies of peptides that interact with lipid bilayers, enabling comparison of the results of this work with studies of other antimicrobial peptides and membrane-perturbing molecules (31)(32)(33)(34)(35)(36)(37)(38)(39), thus allowing insight into the mechanism of membrane disruption and the similarities and differences exhibited by the various cationic, amphipathic, R-helical peptides. It also provides a good baseline for comparison when the bilayer composition is varied.…”