Ulla I. M. Gerling

Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Fluorinated amino acids in amyloid formation: a symphony of size, hydrophobicity and α-helix propensity

Fluorinated amino acids can have dramatic effects on protein stability and protein–protein interactions due to the unique stereoelectronic properties of fluorine. Previous approaches to assessing their properties have mainly focused on helical systems, even though fluoro-amino acids are known to exhibit lower intrinsic helix propensities than their hydrocarbon analogues. Fluorination of specific β-sheet positions within globular proteins has been shown to have a stabilizing effect, suggesting that fluorinated amino acids may generally be well suitable for modulating non-helical structures. Still, fluorinated amino acids have rarely been studied in amyloid forming peptides, which take on a characteristically high cross-β-sheet content. Here, we examine the substitution of natural amino acids within an amyloid forming model peptide by amino acids that contain different stoichiometries of fluorine in their side chains. This approach enables a systematic evaluation of the impact of fluorine on amyloid formation. We have investigated the impact of size, hydrophobicity and secondary structure propensities of the fluorinated amino acids on the amyloid formation process. The structure of the model peptide is based on an engineered coiled coil folding motif that was designed to provide an α-helical starting structure that can fold into β-sheet rich amyloids under controlled conditions. Substitution with fluorinated amino acids was accomplished for two neighboring valine residues that play a key role in the structural transition. The resulting peptides show an unexpected folding behavior as a consequence of the interplay of stereoelectronic effects, helix propensity, hydrophobicity and position of the particular substitution within the amyloid forming system.

The Amyloid Precursor Protein C-Terminal Fragment C100 Occurs in Monomeric and Dimeric Stable Conformations and Binds γ-Secretase Modulators

The amyloid-β (Aβ) peptide is contained within the C-terminal fragment (β-CTF) of the amyloid precursor protein (APP) and is intimately linked to Alzheimers disease. In vivo, Aβ is generated by sequential cleavage of β-CTF within the γ-secretase module. To investigate γ-secretase function, in vitro assays are in widespread use which require a recombinant β-CTF substrate expressed in bacteria and purified from inclusion bodies, termed C100. So far, little is known about the conformation of C100 under different conditions of purification and refolding. Since C100 dimerization influences the efficiency and specificity of γ-secretase cleavage, it is also of great interest to determine the secondary structure and the oligomeric state of the synthetic substrate as well as the binding properties of small molecules named γ-secretase modulators (GSMs) which we could previously show to modulate APP transmembrane sequence interactions [Richter et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14597-14602]. Here, we use circular dichroism and continuous-wave electron spin resonance measurements to show that C100 purified in a buffer containing SDS at micelle-forming concentrations adopts a highly stable α-helical conformation, in which it shows little tendency to aggregate or to form higher oligomers than dimers. By surface plasmon resonance analysis and molecular modeling we show that the GSM sulindac sulfide binds to C100 and has a preference for C100 dimers.

Structure analysis of an amyloid-forming model peptide by a systematic glycine and proline scan.

The ability to adopt at least two different stable conformations is a common feature of proteins involved in many neurodegenerative diseases. The involved molecules undergo a conformational transition from native, mainly helical states to insoluble amyloid structures that have high β-sheet content. A detailed characterization of the molecular architecture of highly ordered amyloid structures, however, is still challenging. Their intrinsically low solubility and high tendency to aggregate often considerably limits the application of established high-resolution techniques such as NMR and X-ray crystallography. An alternative approach to elucidating the tertiary and quaternary organization within an amyloid fibril is the systematic replacement of residues with amino acids that exhibit special conformational characteristics, such as glycine and proline. Substitutions within the β-sheet-prone sequences of the molecules usually severely affect their ability to form fibrils, whereas incorporation at external loop- and bend-like positions often has only marginal effects. Here we present the characterization of the internal architecture of a de novo designed coiled-coil-based amyloid-forming model peptide by means of a series of systematic single glycine and proline replacements in combination with a set of simple low-resolution methods. The folding and assembly behavior of the substituted peptides was monitored simultaneously using circular dichroism spectroscopy, Thioflavin T fluorescence staining, and transmission electron microscopy. On the basis of the obtained data, we successfully identify characteristic bend and core positions within the peptide sequence and propose a detailed structural model of the internal fibrillar arrangement.

Synthesis of enantiomerically pure (2S,3S)-5,5,5-trifluoroisoleucine and (2R,3S)-5,5,5-trifluoro-allo-isoleucine

Summary A practical route for the stereoselective synthesis of (2S,3S)-5,5,5-trifluoroisoleucine (L-5-F3Ile) and (2R,3S)-5,5,5-trifluoro-allo-isoleucine (D-5-F3-allo-Ile) was developed. The hydrophobicity of L-5-F3Ile was examined and it was incorporated into a model peptide via solid phase peptide synthesis to determine its α-helix propensity. The α-helix propensity of 5-F3Ile is significantly lower than Ile, but surprisingly high when compared with 4’-F3Ile.

Multiple glycosylation of de novo designed α-helical coiled coil peptides

The aim of this study was to investigate the influence of multiple O-glycosylation in alpha-helical coiled coil peptides on the folding and stability. For this purpose we systematically incorporated one to six beta-galactose residues into the solvent exposed positions of a 26 amino acid long coiled coil helix. Surprisingly, circular dichroism spectroscopy showed no unfolding of the coiled coil structure for all glycopeptides. Thermally induced denaturations reveal a successive but relative low destabilization of the coiled coil structure upon introduction of beta-galactose residues. These first results indicate that O-glycosylation of the glycosylated variants is easily tolerated by this structural motif and pave the way for further functional studies.

Concluding the Amyloid Formation Pathway of a Coiled-Coil-Based Peptide from the Size of the Critical Nucleus

The size of the critical nucleus acting as intermediate in the amyloid formation of a model peptide is calculated. The theoretical approach is based on experimentally determined amyloid formation rates and gives new insights into the amyloid formation pathway.