Jochen Buerck

A Novel Dendrimeric Peptide with Antimicrobial Properties: Structure-Function Analysis of SB056

The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic β-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as β-sheets in the presence of anionic vesicles. Lipid monolayer surface pressure experiments revealed unusual kinetics of monolayer penetration, which suggest lipid-induced aggregation as a membranolytic mechanism. NMR analyses of the linear monomer and the dendrimeric SB056 in water and in 30% trifluoroethanol, on the other hand, yielded essentially unstructured conformations, supporting the excellent solubility and storage properties of this compound. However, simulated annealing showed that many residues lie in the β-region of the Ramachandran plot, and molecular-dynamics simulations confirmed the propensity of this peptide to fold as a β-type conformation. The excellent solubility in water and the lipid-induced oligomerization characteristics of this peptide thus shed light on its mechanism of antimicrobial action, which may also be relevant for systems that can form toxic β-amyloid fibrils when in contact with cellular membranes. Functionally, SB056 showed high activity against Gram-negative bacteria and some limited activity against Gram-positive bacteria. Its potency against Gram-negative strains was comparable (on a molar basis) to that of colistin and polymyxin B, with an even broader spectrum of activity than numerous other reference compounds.

Molecular structure and function of myelin protein P0 in membrane stacking

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in fluidity changes of the bilayers themselves, but also potentially involves the rearrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

Membrane Fusion is Induced by Antimicrobial and Cell Penetrating Peptides, to an Extent that Correlates with their Conformational Change

Antimicrobial peptides (AMPs) kill bacteria via membrane permeabilization, whereas cell penetrating peptides (CPPs) can cross cellular membranes without causing damage. Yet, many AMPs and CPPs resemble one another, being short cationic peptides, which tend to be unfolded in solution but assume some kind of amphiphilic structure in the membrane-bound state. Fusogenic peptides (FPs) represent a third functional class, responsible e.g. for viral infection, and they are described as short and hydrophobic sequences with a pronounced conformational plasticity.Despite their distinctly different biological roles, we have tested the ability of all three classes of membrane-active peptides to trigger membrane fusion. The HIV1 fusion peptide FP23 is used as a reference to compare the fusion activities of several representative AMPs and CPPs with different conformational preferences and compositions. A fluorescence dequenching assay was used to monitor lipid mixing, and dynamic light scattering revealed the size-increase of the fused vesicles. Several AMPs and CPPs were thus found to be fusogenic to an even higher degree than FP23, which had not been expected. Some insight into the reason for this remarkable activity was obtained by monitoring the secondary structure of the peptides in aqueous buffer before, and in the membrane-bound state after fusion. We found a correlation between the extent of fusion and the extent of lipid-induced folding, suggesting that the energy released in the conformational change is responsible for perturbing the lipid packing in the bilayer and thereby triggering fusion.