Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril

Abstract

The molecular conformation of peptide fragment 105-115 of transthyretin, TTR(105-115), previously shown to form amyloid fibrils in vitro, has been determined by magic-angle spinning solid-state NMR spectroscopy. 13C and 15N linewidth measurements indicate that TTR(105-115) forms a highly ordered structure with each amino acid in a unique environment. 2D 13C-13C and 15N-13C-13C chemical shift correlation experiments, performed on three fibril samples uniformly 13C,15N-labeled in consecutive stretches of 4 aa, allowed the complete sequence-specific backbone and side-chain 13C and 15N resonance assignments to be obtained for residues 105-114. Analysis of the 15N, 13CO, 13Calpha, and 13Cbeta chemical shifts allowed quantitative predictions to be made for the backbone torsion angles phi and psi. Furthermore, four backbone 13C-15N distances were determined in two selectively 13C,15N-labeled fibril samples by using rotational-echo double-resonance NMR. The results show that TTR(105-115) adopts an extended beta-strand conformation that is similar to that found in the native protein except for substantial differences in the vicinity of the proline residue.

Fig 1.

Amino acid sequence of TTR(105–115) and labeling scheme used for the resonance assignments experiments. (U-¹³C,¹⁵N)-labeled residues are underlined.

Fig 2.

Electron micrograph of TTR(105–115) fibrils. Amyloid fibrils were diluted to 100–200 μg/ml and negatively stained by using 2% (wt/vol) uranyl acetate. Fibrils were viewed in a Jeol 1200EX transmission electron microscope, using an accelerating voltage of 80 kV. (Scale bar: 200 nm.)

Fig 3.

1D ¹³C and ¹⁵N MAS NMR spectra of (U-¹³C,¹⁵N)-labeled TTR(105–115) fibrils. ¹³C carbonyl and aromatic (a, d, and g), ¹³C aliphatic (b, e, and h), and ¹⁵N amide (c, f, and i) spectral regions are shown for the TTR(105–115)_YTIA (a–c), TTR(105–115)_AALL (d–f), and TTR(105–115)_LSPY (g–i) samples. ¹³C and ¹⁵N spectra were acquired by using the ramped CP pulse sequence (see text) with 128 and 512 scans, respectively, a 3-s recycle delay, and spinning frequencies of 8.929 kHz (a–c), 10.87 kHz (d–f), and 10.0 kHz (g–i).

Fig 4.

2D ¹³C-¹³C correlation spectrum of TTR(105–115)_YTIA fibrils. The data were acquired at the spinning frequency of 8.929 kHz by using the proton-driven spin diffusion pulse sequence with a 10-ms ¹³C-¹³C mixing time (see text). The 2D data set was acquired according to Ruben and coworkers (36), with 200 complex points in the indirect dimension and the increment of 40 μs, resulting in the total t₁ evolution time of 8 ms. Sixty-four transients were averaged per point with a 3-s recycle delay, resulting in the total 2D acquisition time of ≈22 h. The intraresidue one-bond correlations are indicated by colored dotted lines as follows: Y105, blue; T106, red; I107, green; and A108, indigo.

Fig 5.

2D ¹⁵N-¹³C-¹³C correlation spectra of TTR(105–115)_YTIA fibrils. The NCOCX spectrum (a) correlates the ¹⁵N_i and ¹³C_i-1 resonances, and the NCACX spectrum (b) correlates the ¹⁵N_i and ¹³C_i resonances. The data were acquired by using the NCOCX and NCACX pulse sequences (see text), with a 3-ms ¹⁵N-¹³CO or ¹⁵N-C^α band-selective CP transfer followed by a 10-ms ¹H-driven spin-diffusion ¹³C-¹³C mixing, and the spinning frequency of 8.929 kHz. The 2D data set was acquired according to Ruben and coworkers (36), with 21 complex points in the indirect dimension and the increment of 0.4 ms, resulting in the total t₁ evolution time of 8 ms. A total of 256 transients were averaged per point and a 3-s recycle delay was used, resulting in the total 2D acquisition time of ≈10 h. The residues participating in the ¹⁵N-¹³C correlations are labeled as follows: Y105, blue; T106, red; I107, green; and A108, indigo.

Fig 6.

Secondary ¹⁵N and ¹³C chemical shifts in TTR(105–115) fibrils. The secondary shifts (Δδ) for amide ¹⁵N (a), ¹³C carbonyl (b), ¹³C^α (c), and ¹³C^β (d) resonances were calculated as Δδ = δ_EXP − δ_RC, where δ_EXP and δ_RC are the experimentally observed and random coil chemical shifts in ppm, respectively. The random coil shifts correspond to the values used by the talos program (39) to provide quantitative predictions for the torsion angles φ and ψ (compare Table 2). The secondary shifts for Y105 ¹⁵N, S115 ¹⁵N, S115 ¹³CO, and S115 ¹³C^β could not be calculated; Y105 is the N-terminal residue and S115 was not (U-¹³C,¹⁵N) labeled in any of the samples (S115 ¹³C^α chemical shift was obtained by using a selectively labeled sample).

Fig 7.

Backbone ¹³C-¹⁵N distances measured in selectively labeled TTR(105–115) fibrils by using REDOR. The schematic of the peptide backbone and the specific distances measured are shown (a). Distances indicated in a as 1 and 2 were measured in fibrils isotopically labeled at A108 ¹³CO, L111 ¹³C^α, and L110 ¹⁵N and distances indicated as 3 and 4 were measured in fibrils labeled at S112 ¹³CO, S115 ¹³C^α, and Y114 ¹⁵N. Experimental (circles) and simulated (lines) REDOR S/S₀ curves are shown (b). The experimental curves correspond to the distances: A108 CO-L110 N (), L111 C^α-L110 N (○), and S112 CO-Y114 N (•). The measured distances are indicated in b and summarized in Table 3 (the S115 C^α-Y114 N measurement was omitted for clarity). Spectra were acquired with 320–384 transients and the spinning frequency of 10.0 kHz ± 5 Hz.

Fig 8.

X-ray structure of the peptide fragment corresponding to residues 105–115 in WT TTR (13) (Upper) and the backbone model for the TTR(105–115) peptide in the fibrillar state constructed by using the φ and ψ angles in Table 2 (Lower). The Y105 and T106 backbone torsion angles used in the model were obtained from SSNMR measurements of internuclear distances and torsion angles (unpublished data) analogous to those published in refs. . No constraints are available for ψ_S115 and the experiments designed to probe side-chain χ angles are currently in progress (unpublished data). These moieties are present for illustration purposes only and their conformations in the model correspond to those found in WT TTR (13). The figure was prepared by using the program insight ii version 2000 (Accelerys, San Diego).