A recently proposed lipid-chaperone hypothesis suggests that free lipid molecules, not bound to membranes, affect the aggregation of amyloidogenic peptides such as amyloid-β (Aβ) peptides, whose aggregates are the hallmarks of Alzheimer's disease. Here, we combine experiments with all-atom molecular dynamics simulations in explicit solvent to explore the effects of neuronal ganglioside GM1, abundant in mammalian brains, on the aggregation of two principal isoforms of Aβ, Aβ40 and Aβ42. Our simulations show that free GM1 forms stable, highly water-soluble complexes with both isoforms, and nuclear magnetic resonance experiments support the formation of well-ordered, structurally compact GM1+Aβ complexes. By simulation, we also show that Aβ40 monomers display a preference for binding to GM1-containing heterooligomers over GM1-lacking homo-oligomers, while Aβ42 monomers have the opposite preference. These observations explain why GM1 dose-dependently inhibits Aβ40 aggregation but has no effect on Aβ42 aggregation, as assessed by thioflavin T fluorescence.
Structural studies of membrane proteins in native-like environments require the development of diverse membrane mimetics. Currently there is a need for nanodiscs formed with nonionic belt molecules to avoid nonphysiological electrostatic interactions between the membrane system and protein of interest. Here, we describe the formation of lipid nanodiscs from the phospholipid DMPC and a class of nonionic glycoside natural products called saponins. The morphology, surface characteristics, and magnetic alignment properties of the saponin nanodiscs were characterized by light scattering and solid-state NMR experiments. We determined that preparing nanodiscs with high saponin/lipid ratios reduced their size, diminished their ability to spontaneously align in a magnetic field, and favored insertion of individual saponin molecules in the lipid bilayer surface. Further, purification of saponin nanodiscs allowed flipping of the orientation of aligned nanodiscs by 90°. Finally, we found that aligned saponin nanodiscs provide a sufficient alignment medium to allow the measurement of residual dipolar couplings (RDCs) in aqueous cytochrome c.
Poly(A)-binding protein (Pab1 in yeast) is a canonical stress granule marker. Upon cellular stress including heat shock and starvation, Pab1 phase separates, demixing from the cytosol into foci in vivo. Similarly in vitro, the protein phase separates at heat shock temperatures (above 40 C) and at acidic pHs that occur during other stresses including starvation. Pab1 contains four highly charged RNA recognition motifs (RRMs), connected by linkers, followed by an intrinsically disordered proline-rich (P-) domain and a C-terminal peptide binding domain. We have shown that Pab1 demixing during stress is an adaptive response that can be modulated by hydrophobic mutations in its intrinsically disordered P-domain region (Riback et al (2017) Cell 168:1028. However, the role of Pab1's RRMs, which are necessary and sufficient for temperature demixing in vitro and in vivo, remain unclear. Here we further elucidate the molecular mechanism of Pab1's RRMs on the pH dependence of T demix , the demixing temperature. Since T demix drops rapidly as pH is lowered below physiological pH, we investigated the role of Pab1's 8 histidines, located in its RRMs, by substituting them with the consensus residues in the Pab1 family. Variants having multiple histidines replaced in a single RRM domain exhibit a slightly elevated T demix at all pH values. The variant missing all 8 histidines exhibits a greatly diminished pH sensitivity with its T demix remaining nearly constant, effectively abolishing the pH dependence of demixing within a physiological range. Overall, we find that T demix is proportional to net charge, whether positive or negative, regardless of the substituted positions. Additional factors in the demixing process will be discussed, including linker flexibility and stacking of the RRMs. Together, our studies address how cellular stress is transduced through Pab1's structure to promote its phase separation. Over the last 15 years short peptides have emerged as low molecular weight gelators in water and other solvents. Among them the dipeptide phenylalanylphenylalanine (FF) and its derivatives have attracted considerable attention owing to its capability to self-assemble into a variety of supramolecular structures like nanotubes and even gels. Several lines of evidence suggest that ppinteractions between the phenyl side chains might be the decisive driving force. This notion is further corroborated by the even larger self-assembly propensity of FmocFF where the N-terminal group is substituted by fluorenylmethoxycarbonyl. It is well established that this peptide can become a gelator, if it is first dissolved in dimethylsulfoxide (DMSO) and subsequently mixed with water. We wondered whether FmocFF could already start to aggregate (e.g. into oligomers) in DMSO. We therefore measured the IR, Raman, vibrational circular dichroism (VCD) and proton NMR spectra of FmocFF as a function of peptide concentration and temperature. From the 3 J(N H N a ) constants of the two peptide groups we infer that the peptide predominantly adopt a very...
Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability in standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human Type-2 Diabetes-associated islet amyloid polypeptide (hIAPP, or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant beta-sheet content and the capability of seeding hIAPP fibrillation. We used MAS NMR to demonstrate that the nature of the species was sensitive to sample conditions including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.
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