An unusual peptide conformation may precipitate amyloid formation in Alzheimer's disease: application of solid-state NMR to the determination of protein secondary structure

Abstract

The formation of insoluble proteinaceous deposits is characteristic of many diseases which are collectively known as amyloidosis. There is very little molecular-level structural information available regarding the amyloid deposits due to the fact that the constituent proteins are insoluble and noncrystalline. Therefore, traditional protein structure determination methods such as solution NMR and X-ray crystallography are not applicable. We report herein the application of the solid-state NMR technique rotational resonance (R2) to the accurate measurement of carbon-to-carbon distances in the amyloid formed from a synthetic fragment (H2N-LeuMetValGlyGlyValValIleAla-CO2H) of the amyloid-forming protein of Alzheimer's disease (AD). This sequence has been implicated in the initiation of amyloid formation. Two distances measured by R2 indicate that an unusual structure, probably involving a cis amide bond, is present in the aggregated peptide amyloid. This structure is incompatible with the accepted models of fibril structure. A relationship between this structure and the stability of the amyloid is proposed.

Solid-State NMR as a Probe of Amyloid Structure

“…The first solid state NMR studies of amyloid fibrils were performed by Griffin, Lansbury, and coworkers [4,[17][18][19]. In particular, they applied dipolar recoupling techniques to samples of Aβ [34][35][36][37][38][39][40][41][42] fibrils (where Aβ n-m represents residues n through m of the full-length β-amyloid sequence) prepared with pairs of 13 C labels and obtained constraints on both the peptide conformation and the intermolecular alignment within the β-sheets of these fibrils.…”

“…Stimulated by these earlier results, my laboratory has undertaken solid state NMR studies of fibrils formed by the full-length β-amyloid peptides Aβ 1-40 [9,12,[22][23][24]56] and Aβ 1-42 [11], and by the fragments Aβ [16][17][18][19][20][21][22] [10,13] and Aβ [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] [13,14], employing all of the techniques and structural parameters discussed above. Constraints available in 2002 were used to develop the structural model for Aβ 1-40 fibrils [1,2,22] depicted in Figure 1.…”

“…The strongest evidence for this is the observation of 13 C and 15 N linewidths in the 0.5-1.5 ppm range under MAS for TTR 105-115 and Aβ 11-25 fibrils [14,21]. X-ray diffraction and cryo-EM studies of Aβ [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] fibrils also indicate a very high level of structural order [82,83]. In fibrils formed by longer peptides such as Aβ , structurally ordered segments coexist with disordered segments [9,22,24].…”

“…Thus, structurally ordered and disordered segments within an amyloid fibril can be determined from solid state NMR spectra [9,22]; (3) Nuclear magnetic dipole-dipole couplings depend on interatomic distances as 1/R 3 , allowing distances between specific pairs of carbon and/or nitrogen atoms to be determined, up to a limit of about 0.6 nm and with a precision of approximately 10%. Such distance measurements, which can constrain supramolecular organization [4][5][6][7][8][9][10][11][12][13]15,16] or molecular conformation [6,8,[17][18][19][21][22][23], can be carried out on samples with labels at specific sites [4][5][6][7][8][9][10][11][12][15][16][17][18][19]23] or with multiple uniformly labeled residues [13,21,22] (4) Quantitative constraints on backbone and sidechain torsion angles can be obtained from ″tensor correlation″ techniques, i.e., solid state NMR measurements that are sensitive to the relative orientations of two chemical functional groups and/or bond directions [19,[35][36]…”

“…Solid state NMR, meaning a class of NMR methods developed specifically for structural studies of biochemical (and other) systems in noncrystalline solid and solid-like states, has emerged as a principal methodology for structural studies of amyloid fibrils in the past decade [1][2][3]. Solid state NMR measurements have provided answers to central questions concerning the supramolecular organization of amyloid fibrils [4][5][6][7][8][9][10][11][12][13][14][15][16], peptide conformations within amyloid fibrils [6,8,10,11,[17][18][19][20][21][22][23], and the diversity of amyloid structures [10][11][12][13][14][15]24]. Solid state NMR data have been used as the primary basis for experimentally-constrained structural models of amyloid fibrils, including fibrils formed by the 40-residue β-amyloid (Aβ ) peptide associated with Alzheimer's disease [11,22] and by shorter peptides derived from Aβ or other amyloid-forming proteins [4,5,7,8,10,14,16,…”