Structure Analysis of an Amyloid-Forming Model Peptide by a Systematic Glycine and Proline Scan

Abstract

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.

Inhibition of peptide aggregation by means of enzymatic phosphorylation

“…Phosphorylation experiments were also transferred to model peptide systems by our group. We explored a 26-residue coiled-coil peptide which undergoes a conformational transition to amyloid fibrils in 24 hours under physiological conditions [41], but remains random coil if one of three serine residues carries a phosphate group [27]. The aggregation process could be restored by addition of Lambda Protein Phosphatase that removes the phosphate group [42].…”

The role of proline‐containing peptide triads in β‐sheet formation: A kinetic study

“…Nonetheless, the influence of proline on the folding of even‐numbered strands remained elusive in studies of this new generation of polypeptides. Strategic proline substitutions in short 26‐residue, amyloid‐forming model peptides have been investigated and characterized by circular dichroism (CD) spectroscopy and thioflavin T (ThT) fluorescence staining …”

“…Strategic proline substitutions in short 26-residue, amyloid-forming model peptides have been investigated and characterized by circular dichroism (CD) spectroscopy and thioflavin T (ThT) fluorescence staining. 24,31 Selective single and double proline substitutions at turns proximal to the N-and C-termini of an 11-kDa (143-residue) de novo designed polypeptide, YE8, GH 6 -[(GA) 3 GY(GA) 3 GE] 8 -GAH 6 influence the rate of folding. Glycylalanyl diad (GA) 3 repeats were selected for the formation of identical, weakly interacting b-strands (an even number of residues per b-strand), whereas aromatic amino acids and/or charged amino acid residues, e.g., Y and E, respectively, which are sensitive to environmental changes, were chosen for the c-turn construct.…”

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

“…A nucleus consisting of oligomeric aggregates can also be formed by native or native-like structures, and the conversion to amyloids subsequently takes place within the aggregates. [36][37][38] Here we calculated the nucleus size of a model peptide that has been previously established and well characterized in our group, [46][47][48][49][50][51][52][53] VW18, by applying the nucleated growth polymerization theory to experimental data. A wide range of experimental strategies is currently being developed to detect the actual nucleus size.…”

“…[64] This partial b-sheet conformation could enable the attachment of further peptide strands by the formation of hydrogen bonds. [48] We have presented a theoretical approach that allows calculating the size of the critical nucleus (a necessary intermediate in the aggregation pathway) from experimentally determined amyloid formation rates. [47] The preference for the final three-stranded bsheet conformation could also emerge at a stage where several peptide strands are already attached to each other during the process of oligomerization.…”