Mechanism of amyloid β-protein dimerization determined using single-molecule AFM force spectroscopy.

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments

Mechanism of amyloid β-protein dimerization determined using single-molecule AFM force spectroscopy.

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments

C-Terminal Turn Stability Determines Assembly Differences between Aβ40 and Aβ42

Abstract Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36-Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31-Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31-35 and residues 38-42. In contrast, Aβ(31-40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val-Val36Pro-Gly38Val ("VPV") facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution D-Pro36-L-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36-Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target

Structural stability and refolding of the soybean Kunitz trypsin inhibitor

Soybean Kunitz trypsin inhibitor (SKTI) is a minor allergen found in foods containing soy proteins. Apart from being a food allergen, it has also played a key role in elucidating the mechanism of protease-protease inhibitor interactions that has helped in our better understanding of the action of proteases. SKTI has two well-conserved disulfide bridges that play an important part in its structure. Proteolysis studies on reduced SKTI were performed with different reducing agents and its stability assessed in time course experiments. Thermal denaturation studies were done on SKTI due to its refractoriness to conventional chemical denaturants and also since foods generally undergo heat treatment and processing. During the process of thermal denauration at both controlled and different rates, the different conformations of SKTI were probed using proteases like chymotrypsin and pepsin. This was done to assess their similarity with the native conformation that is protease resistant. Conformational transitions during thermal denaturation were monitored using 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence, intrinsic tryptophan fluorescence, CD and UV absorbance spectroscopy. Structure-function relation studies were also done on SKTI during thermal denaturation of native and reduced inhibitor. Acid denaturation studies were done on SKTI to mimic the gastrointestinal environment that these food allergens are routinely exposed to. Structural transitions in SKTI were monitored in Simulated Gastric Fluid and in HCl-water mixtures using far and near UV CD and ANS fluorescence spectroscopy. The research presented here represents a protein structural stability study of an allergen like SKTI. Hence in the context of protein structure and folding, its allergenic potential is discussed