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.

Proline-glutamate chimera's side chain conformation directs the type of ß-hairpin structure.

Our aim was to study the impact of two proline chimeras, containing a glutamic acid side chain in cis- or trans-configuration, on secondary structure formation. We further investigated to what extent the configuration of the side chain contributes to the overall peptide conformation. We used a 10 residue peptide (IYSNPDGTWT) that forms a ß-hairpin in water. The turn-forming proline was substituted with either a cis- or trans-proline-glutamic acid chimera, resulting in the peptides IYSNPcis -E DGTWT (P1_Pcis-E) and IYSNP(trans-E)DGTWT (P1_Ptrans-E). We studied the conformation of the modified peptides by circular dichroism (CD) and NMR-spectroscopy, and SEC/static light scattering (SLS) analysis. NMR analysis reveals that the modified peptides maintain the ß-hairpin conformation in aqueous solution. At 5 °C and pH 4.3, the peptide (P1_Pcis-E) was found to adopt two coexisting ß-hairpin conformations (2:2 ß-hairpin, and 3:5 ß-hairpin). In contrast to that, the peptide (P1_Ptrans-E) adopts a 2:2 ß-hairpin that exists in equilibrium with a 4:4 ß-hairpin conformation. The adoption of ordered ß-hairpin structures for both modified peptides could be confirmed by CD spectroscopy, while SEC/SLS analysis showed a monomeric oligomerization state for all three investigated peptides. With the combination of several NMR methods, we were able to elucidate that even small alterations in the side chain conformation of the proline-glutamate chimera (cis or trans) can significantly influence the conformation of the adopted ß-hairpin.

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

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.

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.

Conjugate hydrotrifluoromethylation of α,ß-unsaturated acyl-oxazolidinones: synthesis of chiral fluorinated amino acids.

A novel conjugate hydrofluoroalkylation of α,ß-unsaturated acyl-oxazolidinones is described. Using this method, enantiomerically pure ß-trifluoromethylated amino acids were prepared. Trifluorovaline and trifluoroisoleucine were incorporated into peptides and found to show extremely low α-helix propensities.

Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions.

Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references).

Inhibition of amyloid aggregation by formation of helical assemblies.

The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-ß-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a ß-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a ß-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.

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 ß-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 ß-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.

A systematic study of fundamentals in α-helical coiled coil mimicry by alternating sequences of ß- and γ-amino acids.

Aimed at understanding the crucially important structural features for the integrity of α-helical mimicry by ßγ-sequences, an α-amino acid sequence in a native peptide was substituted by differently arranged ßγ-sequences. The self- and hetero-assembly of a series of αßγ-chimeric sequences based on a 33-residue GCN4-derived peptide was investigated by means of molecular dynamics, circular dichroism, and a disulfide exchange assay. Despite the native-like behavior of ßγ alternating sequences such as retention of α-helix dipole and the formation of 13-membered α-helix turns, the αßγ-chimeras with different ßγ substitution patterns do not equally mimic the structural behavior of the native parent peptide in solution. The preservation of the key residue contacts such as van der Waals interactions and intrahelical H-bonding, which can be met only by particular substitution patterns, thermodynamically favor the adoption of coiled coil folding motif. In this study, we show how successfully the destabilizing structural consequences of α â†’ ßγ modification can be harnessed by reducing the solvent-exposed hydrophobic surface area and placing of suitably long and bulky helix-forming side chains at the hydrophobic core. The pairing of αßγ-chimeric sequences with the native wild-type are thermodynamically allowed in the case of ideal arrangement of ß- and γ-residues. This indicates a similarity in local side chain packing of ß- and γ-amino acids at the helical interface of αßγ-chimeras and the native α-peptide. Consequently, the backbone extended residues are able to participate in classical "knob-into-hole" packing with native α-peptide.

The amyloid precursor protein C-terminal fragment C100 occurs in monomeric and dimeric stable conformations and binds γ-secretase modulators.

The amyloid-ß (Aß) peptide is contained within the C-terminal fragment (ß-CTF) of the amyloid precursor protein (APP) and is intimately linked to Alzheimer's disease. In vivo, Aß is generated by sequential cleavage of ß-CTF within the γ-secretase module. To investigate γ-secretase function, in vitro assays are in widespread use which require a recombinant ß-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.

Multiple glycosylation of de novo designed alpha-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.