Amyloid fibrils are self-assembled, macromolecular aggregates formed by peptides and proteins, and associated with numerous diseases including Alzheimer's, Parkinson's, Type II diabetes, senile systemic amyloidosis, etc. A mechanism for formation of these fibrils involves a partially folded state that leads to self-association of the protein into an amyloidogenic intermediate state, which provides a template for the further deposition, and results in amyloid fibril formation. Amyloid fibrils typically exhibit three defining characteristics: (i) exhibit long unbranched fibrils in electron micrographs of ~100 Å diameter (Figure 1A), (ii) show the cross-β X-ray diffraction pattern with lines at ~4.7 Å and ~8-10 Å for strand-strand and sheet-sheet distances, respectively, (Figure 1B) and (iii), they bind Congo red and/or thioflavin-T respectively. While all fibrils have a core β-sheet structure, they can possess other structural elements such as loops and termini that exhibit various degrees of mobility. X-ray diffraction and solution NMR, the primary tools of structural biology, are not applicable to these systems, therefore, a critical barrier to progress in this field is the availability of a general approach to provide structures of amyloid fibrils.
Left: Background image of triplet TTR(105-115) fibril taken using TEM (Left). TTR(105-115) triplet fibril fitted into cryo-EM reconstruction (Center and Right). Right: TEM of TTR(105-115) fibrils showing doublet, triplet and quadruplet fibrils (Background). 3D reconstruction of the doublet (foreground left), triplet (foreground center), and quadruplet (foreground right) fibrils. (from A.W. Fitzpatrick, et al. Proc. Natl. Acad. Sci 110, 5468-5473 (2013) DOI: 10.1073/pnas.1219476110)
Magic Angle Spinning (MAS) NMR is able to yield isotropic spectra, and dipolar recoupling experiments yield distance constraints, making MAS NMR the clear choice to determine the atomic level structure of amyloid fibrils. The Griffin group has developed and utilizes a variety of homo- and hetero-nuclear dipolar recoupling sequences to obtain structural information on amyloid fibrils. Experiments include RFDR, TEDOR, PAR, PAIN, PDSD, DARR. Currently we are utilizing 1H detection to obtain longer-distance structural constraints.
We employ sparsely labeled samples to alleviate spectral congestion and residual J-coupling yielding narrow linewidths and helping to produce complete atomic level structures. We typically utilize 1,3 glycerol, 2-glycerol, 1,6,glucose, and 2-glucose.
We are currently interested in determining the structure of amyloid fibrils of PI3-SH3, β-2microglobulin and the corresponding ΔN-6 variant.