Hetero-assembly of a dual ß-amyloid variant peptide system.

Self-assembly of amyloid polypeptides (1) imparts biological effects depending on the size in over 20 amyloid diseases and (2) produces useful yet relatively untapped biomaterials. Unfortunately, our understanding of amyloid polypeptides, as related to biomedical implications and biomaterial applications, is limited by their self-assembling nature. In this study, we report the creation of a dual peptide system, where a pair of ß-amyloid (Aß) variants are not self-assembled but hetero-assembled in the presence of their assembly partners. We provide evidence that the resulting hetero-assemblies share molecular, structural and morphological similarities with typical amyloid self-assemblies formed by a single polypeptide (e.g., Aß). We anticipate that our dual peptide system may readily be adapted for precise control of amyloid assembly, for the study of size-dependent neurotoxicity and precise fabrication of amyloid biomaterials.

Molecular Determinants for Protein Stabilization by Insertional Fusion to a Thermophilic Host Protein.

A universal method that improves protein stability and evolution has thus far eluded discovery. Recently, however, studies have shown that insertional fusion to a protein chaperone stabilized various target proteins with minimal negative effects. The improved stability was derived from insertion into a hyperthermophilic protein, Pyrococcus furiosus maltodextrin-binding protein (PfMBP), rather than from changes to the target protein sequence. In this report, by evaluating the thermodynamic and kinetic stability of various inserted ß-lactamase (BLA) homologues, we were able to examine the molecular determinants of stability realized by insertional fusion to PfMBP. Results indicated that enhanced stability and suppressed aggregation of BLA stemmed from enthalpic and entropic mechanisms. This report also suggests that insertional fusion to a stable protein scaffold has the potential to be a useful method for improving protein stability, as well as functional protein evolution.

Engineering of a peptide probe for ß-amyloid aggregates.

Aggregation of ß-amyloid (Aß) is central to the pathogenesis of Alzheimer's disease (AD). Aß aggregation produces amyloid assemblies, such as oligomers and fibrils. In contrast to non-toxic Aß monomers, Aß oligomers and fibrils can act directly as major toxic agents and indirectly as pools of the toxic entities, respectively. Thus, the detection of Aß aggregates is of diagnostic interest and should benefit enhanced molecular understanding of AD. Among many molecular platforms, peptide-based ligands hold promise as Aß probes due to their relative simplicity, ease of optimization and facile conjugation to other molecular contexts. In this regard, Aß hydrophobic segments (critical in Aß self-assembly) or variants thereof can serve as lead molecules for Aß probe development. Unfortunately, the resulting peptides are either highly self-aggregation-prone or their probe potential has not been thoroughly examined. In the present study, we characterized a novel peptide ligand, KLVFWAK, which was created by simple point mutations of an Aß hydrophobic segment ((16)KLVFFAE(22)). We found that KLVFWAK displayed low self-aggregation propensity and was preferentially bound to Aß oligomers and fibrils relative to Aß monomers. Interestingly, binding of KLVFWAK to Aß aggregates occurred at a non-homologous Aß segment (e.g., Aß C-terminal domain) rather than the homologous (16)KLVFFAE(22). We also show that detection of Aß aggregates during incubation of fresh Aß was possible with KLVFWAK, further supporting KLVFWAK's high probe potential for Aß aggregates. In short, this study presents creation of a non-self-aggregating peptide ligand for Aß aggregates through simple point mutation of an Aß-derived segment.