Henriette Mozsolits

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance.

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association (k(a)) and dissociation (k(d)) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl-L-alpha-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high alpha-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.

Surface plasmon resonance analysis of antimicrobial peptide-membrane interactions: affinity & mechanism of action

Antimicrobial peptides are being increasingly recognised as potential candidates for antibacterial drugs in the face of the rapidly emerging bacterial resistance to conventional antibiotics in recent years. However, a precise understanding of the relationship between antimicrobial peptide structure and their cytolytic function in a range of organisms is still lacking. This is a result of the complex nature of the interactions of antimicrobial peptides with the cell membrane, the mechanism of which can vary considerably between different classes of antimicrobial peptides. A wide range of biophysical techniques have been used to study the influence of a number of peptide and membrane properties on the cytolytic activity of these peptides model membrane systems. Until recently, however, very few studies had reported measurements of the affinity of antimicrobial peptides for different membrane systems mainly due to the difficulty in obtaining this information. Surface plasmon resonance (SPR) spectroscopy has recently been applied to the study of biomembrane-based systems which has allowed a real-time analysis of binding affinity and kinetics. This mini review provides an overview of the recent applications that demonstrate the potential of SPR to study the membrane interactions of antimicrobial peptides.

The interaction of bioactive peptides with an immobilized phosphatidylcholine monolayer.

The interaction of three bioactive peptides, bombesin, beta-endorphin, and glucagon with a phosphatidylcholine monolayer that was immobilized to porous silica particles and packed into a stainless steel column cartridge, has been studied using dynamic elution techniques. This immobilized lipid monolayer provides a biophysical model system with which to study the binding of peptides to a lipid membrane. In particular, the influence of temperature and methanol concentration on the affinity of each peptide for the immobilized lipid surface was assessed. For all test peptides, nonlinear retention plots were observed at all temperatures that contrasted sharply with the simple linear plots observed for the small unstructured control molecules N-acetyltryptophanamide and diphenylalanine. An analysis of the thermodynamics of the interaction of peptides with the immobilized monolayer was also carried out. The results revealed that while the peptides interacted with the monolayer predominantly through hydrophobic interactions, the relative contribution of DeltaH(assoc)(O) and DeltaS(assoc)(O) to the overall free energy of association was dependent on the temperature and methanol concentration. In particular, it was evident that under most conditions, the binding of the peptides to the immobilized lipid monolayer was enthalpy-driven, i.e., mediated by nonclassical hydrophobic interactions. Significant band-broadening and asymmetric and split peaks were also observed for bombesin, beta-endorphin, and glucagon at different temperatures and methanol concentrations. These changes in affinity and peak shape are consistent with the formation of multiple conformational species during the interaction of these peptides with the lipid monolayer. In addition, the binding behavior of the three test peptides on an n-octylsilica surface that lacked the phospho headgroups of the phospholipid was significantly different from that observed with the immobilized phosphatidylcholine surface, indicating a specificity of interaction between the peptides and the lipid surface. Overall, these experimental results demonstrate that the biomimetic phosphatidylcholine monolayer provides a stable and sensitive system with which to explore the molecular mechanism of peptide conformational changes during membrane interactions.

Tethering of the Proximal Region of the Angiotensin II Receptor (AT1A) C-Terminus to the Cell Membrane

The type 1 angiotensin receptor (AT1A) is a 359 amino acid G protein-coupled receptor that mediates the important cardiovascular and homeostatic actions of the peptide hormone angiotensin II (AngII) [1]. The 54 amino acid carboxyl-terminus (Leu305 to Glu359) of the AT1A receptor interacts with, and activates, G proteins and other signaling molecules, indicating a contribution to receptor activation, while the identification of phosphorylation sites and internalisation motifs suggest a key role in receptor regulation [2]. The C-terminus of the type I angiotensin II receptor (AT1A) is thus a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal region of the C-terminus adopt a helical structure in hydrophobic environments [3], which may relate to the capacity of this region to act as an important switch site in vivo. In order to explore this hypothesis, we examined whether this basic-charged, amphipathic α-helical region can interact with lipid components in the cell membrane and thereby modulate local receptor structure.