Continuous Flow Reactor for the Production of Stable Amyloid Protein Oligomers

The predominant working hypothesis of Alzheimer's disease is that the proximate pathologic agents are oligomers of the amyloid β-protein (Aβ). "Oligomer" is an ill-defined term. Many different types of oligomers have been reported, and they often exist in rapid equilibrium with monomers and higher-order assemblies. This has made formal structure-activity determinations difficult. Recently, Ono et al. [Ono, K., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 14745-14750] used rapid, zero-length, in situ chemical cross-linking to stabilize the oligomer state, allowing the isolation and study of pure populations of oligomers of a specific order (number of Aβ monomers per assembly). This approach was successful but highly laborious and time-consuming, precluding general application of the method. To overcome these difficulties, we developed a "continuous flow reactor" with the ability to produce theoretically unlimited quantities of chemically stabilized Aβ oligomers. We show, in addition to its utility for Aβ, that this method can be applied to a wide range of other amyloid-forming proteins

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

The inverted free energy landscape of an intrinsically disordered peptide by simulations and experiments

The free energy landscape theory has been very successful in rationalizing the folding behaviour of globular proteins, as this representation provides intuitive information on the number of states involved in the folding process, their populations and pathways of interconversion. We extend here this formalism to the case of the A\u3b240 peptide, a 40-residue intrinsically disordered protein fragment associated with Alzheimer's disease. By using an advanced sampling technique that enables free energy calculations to reach convergence also in the case of highly disordered states of proteins, we provide a precise structural characterization of the free energy landscape of this peptide. We find that such landscape has inverted features with respect to those typical of folded proteins. While the global free energy minimum consists of highly disordered structures, higher free energy regions correspond to a large variety of transiently structured conformations with secondary structure elements arranged in several different manners, and are not separated from each other by sizeable free energy barriers. From this peculiar structure of the free energy landscape we predict that this peptide should become more structured and not only more compact, with increasing temperatures, and we show that this is the case through a series of biophysical measurements

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

Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder characterized by amyloid deposition in the cerebral neuropil and vasculature. These amyloid deposits comprise predominantly fragments and full-length (40 or 42 residue) forms of the amyloid beta-protein (Abeta) organized into fibrillar assemblies. Compelling evidence indicates that factors that increase overall Abeta production or the ratio of longer to shorter forms, or which facilitate deposition or inhibit elimination of amyloid deposits, cause AD or are risk factors for the disease. In vitro studies have demonstrated that fibrillar Abeta has potent neurotoxic effects on cultured neurons. In vivo experiments in non-human primates have demonstrated that Abeta fibrils directly cause pathologic changes, including tau hyperphosphorylation. In concert with histologic studies revealing a lack of tissue injury in areas of the neuropil in which non-fibrillar deposits were found, these data suggested that fibril assembly was a prerequisite for Abeta-mediated neurotoxicity in vivo. Recently, however, both in vitro and in vivo studies have revealed that soluble, oligomeric forms of Abeta also have potent neurotoxic activities, and in fact, may be the proximate effectors of the neuronal injury and death occurring in AD. A paradigm shift is thus emerging that necessitates the reevaluation of the relative importance of polymeric (fibrillar) vs. oligomeric assemblies in the pathobiology of AD. In addition to AD, an increasing number of neurodegenerative disorders, including Parkinson's disease, familial British dementia, familial amyloid polyneuropathy, amyotrophic lateral sclerosis, and prion diseases, are associated with abnormal protein assembly processes. The archetypal features of the assembly-dependent neuropathogenetic effects of Abeta may thus be of relevance not only to AD but to these other disorders as well