Dimer Formation Enhances Structural Differences between Amyloid β-Protein (1–40) and (1–42): An Explicit-Solvent Molecular Dynamics Study

Amyloid -protein (A) is central to the pathology of Alzheimer's disease. A 5% difference in the primary structure of the two predominant alloforms, A and A, results in distinct assembly pathways and toxicity properties. Discrete molecular dynamics (DMD) studies of A and A assembly resulted in alloform-specific oligomer size distributions consistent with experimental findings. Here, a large ensemble of DMD–derived A and A monomers and dimers was subjected to fully atomistic molecular dynamics (MD) simulations using the OPLS-AA force field combined with two water models, SPCE and TIP3P. The resulting all-atom conformations were slightly larger, less compact, had similar turn and lower -strand propensities than those predicted by DMD. Fully atomistic A and A monomers populated qualitatively similar free energy landscapes. In contrast, the free energy landscape of A dimers indicated a larger conformational variability in comparison to that of A dimers. A dimers were characterized by an increased flexibility in the N-terminal region D1-R5 and a larger solvent exposure of charged amino acids relative to A dimers. Of the three positively charged amino acids, R5 was the most and K16 the least involved in salt bridge formation. This result was independent of the water model, alloform, and assembly state. Overall, salt bridge propensities increased upon dimer formation. An exception was the salt bridge propensity of K28, which decreased upon formation of A dimers and was significantly lower than in A dimers. The potential relevance of the three positively charged amino acids in mediating the A oligomer toxicity is discussed in the light of available experimental data

Factors That Drive Peptide Assembly and Fibril Formation: Experimental and Theoretical Analysis of Sup35 NNQQNY Mutants

Residue mutations have substantial effects on aggregation kinetics and propensities of amyloid peptides and their aggregate morphologies. Such effects are attributed to conformational transitions accessed by various types of oligomers such as steric zipper or single β-sheet. We have studied the aggregation propensities of six NNQQNY mutants: NVVVVY, NNVVNV, NNVVNY, VIQVVY, NVVQIY, and NVQVVY in water using a combination of ion-mobility mass spectrometry, transmission electron microscopy, atomic force microscopy, and all-atom molecular dynamics simulations. Our data show a strong correlation between the tendency to form early β-sheet oligomers and the subsequent aggregation propensity. Our molecular dynamics simulations indicate that the stability of a steric zipper structure can enhance the propensity for fibril formation. Such stability can be attained by either hydrophobic interactions in the mutant peptide or polar side-chain interdigitations in the wild-type peptide. The overall results display only modest agreement with the aggregation propensity prediction methods such as PASTA, Zyggregator, and RosettaProfile, suggesting the need for better parametrization and model peptides for these algorithms

Preparation of Pure Populations of Amyloid β-Protein Oligomers of Defined Size.

Protein and peptide oligomers are thought to play important roles in the pathogenesis of a number of neurodegenerative diseases. For this reason, considerable effort has been devoted to understanding the oligomerization process and to determining structure-activity relationships among the many types of oligomers that have been described. We discuss here a method for producing pure populations of amyloid β-protein (Aβ) of specific sizes using the most pathologic form of the peptide, Aβ42. This work was necessitated because Aβ oligomerization produces oligomers of many different sizes that are non-covalently associated, which means that dissociation or further assembly may occur. These characteristics preclude rigorous structure-activity determinations. In studies of Aβ40, we have used the method of photo-induced cross-linking of unmodified proteins (PICUP) to produce zero-length carbon-carbon bonds among the monomers comprising each oligomer, thus stabilizing the oligomers. We then isolated pure populations of oligomers by fractionating the oligomers by size using SDS-PAGE and then extracting each population from the stained gel bands. Although this procedure worked well with the shorter Aβ40 peptide, we found that a significant percentage of Aβ42 oligomers had not been stabilized. Here, we discuss a new method capable of yielding stable Aβ42 oligomers of sizes from dimer through dodecamer

Familial Alzheimer’s Disease Mutations Differentially Alter Amyloid β-Protein Oligomerization

[Image: see text] Although most cases of Alzheimer’s disease (AD) are sporadic, ∼5% of cases are genetic in origin. These cases, known as familial Alzheimer’s disease (FAD), are caused by mutations that alter the rate of production or the primary structure of the amyloid β-protein (Aβ). Changes in the primary structure of Aβ alter the peptide’s assembly and toxic activity. Recently, a primary working hypothesis for AD has evolved where causation has been attributed to early, soluble peptide oligomer states. Here we posit that both experimental and pathological differences between FAD-related mutants and wild-type Aβ could be reflected in the early oligomer distributions of these peptides. We use ion mobility-based mass spectrometry to probe the structure and early aggregation states of three mutant forms of Aβ40 and Aβ42: Tottori (D7N), Flemish (A21G), and Arctic (E22G). Our results indicate that the FAD-related amino acid substitutions have no noticeable effect on Aβ monomer cross section, indicating there are no major structural changes in the monomers. However, we observe significant changes to the aggregation states populated by the various Aβ mutants, indicating that structural changes present in the monomers are reflected in the oligomers. Moreover, the early oligomer distributions differ for each mutant, suggesting a possible structural basis for the varied pathogenesis of different forms of FAD

Characterization of peptide-guided polymer assembly at the air/water interface

An organo-soluble, peptide-polymer conjugate that combines poly(n-butyl acrylate) with a beta-sheet-forming peptide is spread at the water surface to investigate peptide-guided self-assembly in a two-dimensional environment. Single layers of the conjugate are studied to gain information on the packing, orientation, and structure of the conjugate molecules using standard monolayer techniques: isotherms, grazing incidence X-ray diffraction (GIXD), and infrared reflection absorption spectroscopy (IRRAS). At all conditions studied, the stabilizing beta-sheet network consists of antiparallel beta-sheets oriented parallel to the air/water interface. The incorporation of temporary switch defects in the peptide segment enables beta-sheet assembly to be triggered at different packing densities. Stable monolayers, with low compressibilities similar to peptide monolayers, form when beta-sheet assembly occurs in monolayers that contain closely packed conjugate molecules. Langmuir-Schaefer transfer of the switched monolayer seeded with 1/1000 part stearic acid results in a transferred monolayer containing ordered domains with 7 nm wide stripes, a width in agreement with the end-to-end distance of the conjugate molecule. In this interfacial system, high packing densities and a hydrophobic seed molecule play an important role in beta-sheet network and structure formation. Both effects likely direct the highly ordered beta-sheet structure because of beta-strand prealignment. Insights gained from self-assembly in this system can be applied to peptide aggregation mechanisms in more complex interfacial environments

Self-Assembly and Hydrogelation of an Amyloid Peptide Fragment

The self-assembly of a fragment of the amyloid β peptide that has been shown to be critical in amyloid fibrillization has been studied in aqueous solution. There are conflicting reports in the literature on the fibrillization of Aβ (16–20), i.e., KLVFF, and our results shed light on this. In dilute solution, self-assembly of NH2−KLVFF−COOH is strongly influenced by aromatic interactions between phenylalanine units, as revealed by UV spectroscopy and circular dichroism. Fourier transform infrared (FTIR) spectroscopy reveals β-sheet features in spectra taken for more concentrated solutions and also dried films. X-ray diffraction and cryo-transmission electron microscopy (cryo-TEM) provide further support for β-sheet amyloid fibril formation. A comparison of cryo-TEM images with those from conventional dried and negatively stained TEM specimens highlights the pronounced effects of sample preparation on the morphology. A comparison of FTIR data for samples in solution and dried samples also highlights the strong effect of drying on the self-assembled structure. In more concentrated phosphate-buffered saline (PBS) solution, gelation of NH2−KLVFF−COOH is observed. This is believed to be caused by screening of the electrostatic charge on the peptide, which enables β sheets to aggregate into a fibrillar gel network. The rheology of the hydrogel is probed, and the structure is investigated by light scattering and small-angle X-ray scattering