John D. Gehman

Effect of antimicrobial peptides from Australian tree frogs on anionic phospholipid membranes.

Skin secretions of numerous Australian tree frogs contain antimicrobial peptides that form part of the host defense mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al. (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as novel antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1, which exist as random coil in aqueous solution but have alpha-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC ( d 54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid headgroups by deuterium and (31)P NMR, respectively. In this report we demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterized using surface plasmon resonance (SPR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides has significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface interaction for aurein 1.2 and pore formation rather than membrane lysis by the longer peptides.

Lipid matrix plays a role in Abeta fibril kinetics and morphology.

Amyloid beta(1–42) binds to Amyloid beta(1–42) by circular dichroism (View interaction) Amyloid beta(1–42) binds to Amyloid beta(1–42) by fluorescence technology (View interaction) Amyloid beta(1–42) binds to Amyloid beta(1–42) by electron microscopy (View interaction).

Metal effects on the membrane interactions of amyloid-β peptides

Aβ(1–42) peptide, found as aggregated species in Alzheimer’s disease brain, is linked to the onset of dementia. We detail results of 31P and 2H solid-state NMR studies of model membranes with Aβ peptides and the effect of metal ions (Cu2+ and Zn2+), which are found concentrated in amyloid plaques. The effects on the lipid bilayer and the peptide structure are different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induce formation of smaller vesicles, but not when Aβ(1–42) is associated with the bilayer membrane. Aβ(25–35), a fragment from the C-terminal end of Aβ(1–42), which lacks the metal coordinating sites found in the full length peptide, is neurotoxic to cortical cortex cell cultures. Addition of metal ions has little effect on membrane bilayers with Aβ(25–35) peptides. 31P magic angle spinning NMR data show that Aβ(1–42) and Aβ(1–42)-Cu2+ complexes interact at the surface of anionic phospholipid membranes. Incorporated peptides, however, appear to disrupt the membrane more severely than associated peptides. Solid-state 13C NMR was used to compare structural changes of Aβ(1–42) to those of Aβ(25–35) in model membrane systems of anionic phospholipids and cholesterol. The Aβ peptides appeared to have an increase in β-strand structure at the C-terminus when added to phospholipid liposomes. The inclusion of Cu2+ also influenced the observed chemical shift of residues from the C-terminal half, providing structural clues for the lipid-associated Aβ/metal complex. The results point to the complex pathway(s) for toxicity of the full-length peptide.

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix-hinge-helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.

Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism.

ABSTRACT Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 μM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.

Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment

Abstract A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH2) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d54-DMPC) and mixed d54-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by 31P NMR. However, no perturbations in the T1 relaxation nor the T2- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).

Self-assembly of peptides into spherical nanoparticles for delivery of hydrophilic moieties to the cytosol

We report a novel class of self-assembling peptide nanoparticles formed by mixing aqueous solutions of K(16) peptide and a 20 amino acid peptide of net charge -5 (GLFEALLELLESLWELLLEA). Particle formation is salt-dependent and yields perfectly spherical nanoparticles of approximately 120 to approximately 800 nm diameter, depending on buffer composition and temperature, with a stoichiometry of approximately 1:2.5 for the cationic and anionic peptides. The anionic peptide forms an alpha-helix in aqueous solution, has all five glutamates on one side of the helix, and exists entirely as a discrete oligomer of 9-10 peptides. A rigid oligomer with 45-50 negative charges almost certainly represents the core component of these nanoparticles, held together by electrostatic interactions with the unstructured K(16) peptide. Cells internalize these particles by an endocytic process, and free particles are frequently seen in the cytosol, presumably because of the acid-dependent fusogenic properties of the anionic peptide. Among other applications, these particles have potential for the targeted delivery of single or multiple therapeutic moieties directly to the cytosol, and we report the successful delivery of a K(16)-linked pro-apoptosis peptide.

Context dependence of protein misfolding and structural strains in neurodegenerative diseases

Vast arrays of structural forms are accessible to simple amyloid peptides and environmental conditions can direct assembly into single phases. These insights are now being applied to the aggregation of the Aβ peptide of Alzheimers disease and the identification of causative phases. We extend use of the imaging agent Pittsburgh compound B to discriminate among Aβ phases and begin to define conditions of relevance to the disease state. Also, we specifically highlight the development of methods for defining the structures of these more complex phases.

Disentanglement of Heterogeneous Dynamics in Mixed Lipid Systems

Static phosphorous NMR has been a powerful technique for the study of model supramolecular phospholipid structures. Application to natural lipid bilayers with complex compositions, however, has been severely limited by the difficulty in deconvoluting overlapping broad lineshapes. We demonstrate a solution to this problem, using a global fit to a few slow magic-angle spinning spectra, in combination with an adaptation of Boltzmann statistics maximum entropy. The method provides a model-free means to characterize a heterogeneous mix of lipid dynamics via a distribution of (31)P chemical shift anisotropies. It is used here to identify clear changes in membrane dynamics of a phosphatidylethanolamine and phosphatidylglycerol mixture, mimicking an Escherichia coli membrane upon addition of just 2% of the antimicrobial peptide maculatin 1.1. This illustration opens the prospect for investigation of arbitrarily complex natural lipid systems, important in many areas of biophysical chemistry and biomedicine.