18 research outputs found
Successive stages of amyloid-? self-assembly characterized by solid-state nuclear magnetic resonance with dynamic nuclear polarization
Self-assembly
of amyloid-β (Aβ) peptides in human brain
tissue leads to neurodegeneration in Alzheimer’s disease (AD).
Amyloid fibrils, whose structures have been extensively characterized
by solid state nuclear magnetic resonance (ssNMR) and other methods,
are the thermodynamic end point of Aβ self-assembly. Oligomeric
and protofibrillar assemblies, whose structures are less well-understood,
are also observed as intermediates in the assembly process in vitro
and have been implicated as important neurotoxic species in AD. We
report experiments in which the structural evolution of 40-residue
Aβ (Aβ40) is monitored by ssNMR measurements on frozen
solutions prepared at four successive stages of the self-assembly
process. Measurements on transient intermediates are enabled by ssNMR
signal enhancements from dynamic nuclear polarization (DNP) at temperatures
below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in
conformational order from an initial state comprised primarily of
monomers and small oligomers in solution at high pH, to larger oligomers
near neutral pH, to metastable protofibrils, and finally to fibrils.
Surprisingly, the predominant molecular conformation, indicated by <sup>13</sup>C NMR chemical shifts and by side chain contacts between
F19 and L34 residues, is qualitatively similar at all stages. However,
the in-register parallel β-sheet supramolecular structure, indicated
by intermolecular <sup>13</sup>C spin polarization transfers, does
not develop before the fibril stage. This work represents the first
application of DNP-enhanced ssNMR to the characterization of peptide
or protein self-assembly intermediates
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Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains
Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification