Fibrils of Truncated Pyroglutamyl-Modified Aβ Peptide Exhibit a Similar Structure as Wildtype Mature Aβ Fibrils

Fibrillation of differently modified amyloid β peptides and deposition as senile plaques are hallmarks of Alzheimer’s disease. N-terminally truncated variants, where the glutamate residue 3 is converted into cyclic pyroglutamate (pGlu), form particularly toxic aggregates. We compare the molecular structure and dynamics of fibrils grown from wildtype Aβ(1–40) and pGlu3-Aβ(3–40) on the single amino acid level. Thioflavin T fluorescence, electron microscopy, and X-ray diffraction reveal the general morphology of the amyloid fibrils. We found good agreement between the 13C and 15N NMR chemical shifts indicative for a similar secondary structure of both fibrils. A well-known interresidual contact between the two β-strands of the Aβ fibrils could be confirmed by the detection of interresidual cross peaks in a 13C-13C NMR correlation spectrum between the side chains of Phe 19 and Leu 34. Small differences in the molecular dynamics of residues in the proximity to the pyroglutamyl-modified N-terminus were observed as measured by DIPSHIFT order parameter experiments.

function of time. In agreement with previous results 6 , it is observed that also under our conditions the fibrillation of pGlu 3 -Aβ (3-40) has a significantly shorter lag time and is overall faster than for WT Aβ . The lag time for pGlu 3 -Aβ (3-40) is 7 ± 1 h, while it is 43 ± 2 h for Aβ . The morphology of the pGlu 3 -Aβ  fibrils was studied by electron microscopy (EM). Figure 1B shows a typical EM micrograph of pGlu 3 -Aβ (3-40) fibrils after 3 weeks of incubation, displaying fibrils of homogeneous morphology. These fibrils have a width of 12.8 ± 2.1 nm (n = 20), which is slightly larger than the mean diameter of WT Aβ (1-40) fibrils (10.0 ± 1.6 nm) 18 . A preferential shorter fibril length for pGlu 3 -Aβ  as reported in 6 could not be observed under our fibrillation conditions. Fibrils of pGlu 3 -Aβ (3-40) exhibit a mean length of 850 ± 300 nm WT Aβ  fibrils of 500 ± 200 nm (n = 30). Also, the X-ray diffraction pattern of pGlu 3 -Aβ (3-40) fibrils (Fig. 1C) exhibit the typical cross-β structure as observed for all other amyloid fibrils. The measured main X-ray reflections correspond to repeat spacings of 4.7 Å and 10.3 Å, which represent the typical values for the interstrand spacing and the intersheet distance, respectively 19 .
To obtain insights into the secondary structure on the level of individual amino acids, solid-state NMR spectra of pGlu 3 -Aβ (3-40) fibrils with uniformly 13 C/ 15 N-labeled amino acids (for the labeling scheme see experimental section) were measured. In choosing the sites for isotopic labeling, we paid special attention to the N-terminus of pGlu 3 -Aβ  to study possible differences in the structures due to the pGlu modification in position 3 and to probe the extent of the known secondary structure elements. To assign the 13 C and 15 N chemical shifts, 13 C-13 C DARR and 15 N-13 Cα NMR correlation spectra were conducted under magic-angle spinning (MAS) conditions in dual acquisition mode 20 . Supplementary Figure S1 shows a 13 C-13 C DARR and a 15 N-13 Cα NMR spectrum as examples. The chemical shifts values for all labeled amino acids are listed in Table S1. Figure 2 reports the 13 Cα and 13 Cβ chemical shifts of pGlu 3 -Aβ  and mature WT Aβ (1-40) fibrils (data taken from ref. 21) as differences from random coil values reported in the literature 22 . Since NMR chemical shifts are sensitive to the secondary structure, values close to zero correspond to random coil regions, while negative values for Cα and positive values for Cβ report β -strand conformations 22 . One can clearly see that most of the chemical shift values of both fibrillar species are very similar. Some alterations are observed for the Cβ signal of Phe 4 , which may result from the direct vicinity to the chemically modified pGlu 3 . For Phe 19 Cβ and Gly 29 Cα , two chemical shift values were observed. Such structural polymorphism of Aβ fibrils has been observed before Overall, a striking structural similarity between pGlu 3 -Aβ (3-40) and WT Aβ (1-40) fibrils is observed and one has to conclude that the typical secondary structure elements of WT Aβ (1-40) with an unstructured N-terminus and two β -strand regions comprising amino acids 10-22 and 30-38, which are connect by a short unstructured region, holds also true for pGlu 3 -Aβ .
For a systematic comparison of the secondary structure of pGlu 3 -Aβ (3-40) fibrils to different preparations of WT Aβ fibrils in different stages of the fibrillation process, Figure 3 shows correlation plots of the differences in the chemical shift values ( 13 Cα -13 Cβ ), which are very sensitive to secondary structure and have the advantage of being independent of chemical shift referencing, which may vary between the different laboratories. On the secondary structure level, a very good correlation of the pGlu 3 -Aβ (3-40) fibrils with the results for WT Aβ fibrils is obtained (A-C). Only slightly smaller correlation coefficients are obtained when pGlu 3 -Aβ  fibrils are compared to protofibrils (D) and oligomers (E, F). This indicates that the secondary structure of the pGlu 3 -Aβ (3-40) fibrils is very similar to mature fibrils, but also to oligomers and protofibrils.
Tertiary structure information for Aβ (1-40) fibrils represents a field of some controversy and is highly dependent on the number and precision of the structural constraints available, which has led to different structural models 16,17,26,27 . To obtain some insight into the spatial relationship of the two β -strands and the tertiary structure of the pGlu 3 -Aβ (3-40) peptides in the fibrils, 13 C-13 C DARR NMR experiments were conducted with a long mixing time of 500 ms. This allows to observe interresidual contacts between carbons in spatial proximity of up to ~6 Å, via the detection of cross peaks between carbons of different amino acids. For mature Aβ (1-40) fibrils as well as oligomers and protofibrils, a contact between the side chains of Phe 19 and Leu 34 has been well-described [16][17][18]28,29 . This contact indicates the close proximity between the two β -strands of the monomer and the U-shaped structure of the monomers in the fibrils. In the DARR NMR spectrum for pGlu 3 -Aβ (3-40) fibrils ( Figure S1), cross peaks between Phe 19 and Leu 34 are clearly visible, especially between the aromatic ring carbons of Phe 19 and the Cβ signal of Leu 34 . This suggests a close structural relationship between pGlu 3 -Aβ (3-40) and WT Aβ (1-40) fibrils also on the tertiary structure level.
It was shown that a molecular contact between Glu 22 and Ile 31 indicates earlier stages of the fibrillation process in oligomers and protofibrils [30][31][32] , but this contact is absent in mature Aβ fibrils 32 . Based on these observations and other data, a model for the reorganization of the hydrogen bonds from intramolecular for oligomers and protofibrils to intermolecular hydrogen bonds for the mature fibrils has been proposed 30,31 . In our 13 C-13 C DARR NMR spectrum of the peptide that contains these two amino acids 13 C/ 15 N-labeled, such a cross peak indicative of the molecular contact was not observed ( Figure S2). This result also confirms that fibrils of pGlu 3 -Aβ (3-40) exhibit a strong structural similarity to mature Aβ (1-40) fibrils.
Solid-state NMR also offers the possibility to investigate the molecular dynamics of the individual segments of the pGlu 3 -Aβ (3-40) fibrils. Such measurements can provide information about the different domains in fibrillar peptides or proteins and substantially support the structural data 21,33,34 . We measured the motionally averaged 1 H-13 C dipolar couplings for each resolved carbon signal in DIPSHIFT experiments and convert these into molecular order parameters. Figure 4 shows the comparison of the order parameters of the backbone Cα of pGlu 3   results in a somewhat higher order parameter, which corresponds to smaller motional amplitudes of the fluctuations of these residues. One further notable exception is the order parameter of Ile 31 , which is significantly higher in pGlu 3 -Aβ (3-40) compared to WT Aβ . This may suggest some importance of this residue for the formation of oligomers and protofibrils as Ile 31 appears to be involved in intramolecular contacts of intermediates, but not in mature fibrils. Interestingly, the correlations of the measured order parameters to the only available datasets for mature Aβ (1-40) fibrils 21 and Aβ (1-40) protofibrils 35 are not as good as observed for the chemical shifts/secondary structure. This may in part reflect the fact that chemical shifts are measured more precisely than motionally averaged dipolar couplings, but could also indicate a dynamic polymorphism that relates to small packing differences of the individual residues in the fibrils.

Conclusion
Overall, we conclude that on the level of the single amino acid, fibrils formed of pGlu 3 -Aβ (3-40) exhibit a strong similarity in the molecular structure compared to WT mature Aβ fibrils. Our data agree with a recent study that reported, on the basis of H/D exchange NMR, FTIR, and CD measurements that modified pGlu 3 -Aβ (3-40) and unmodified Aβ (1-40) comprised similar peptide conformations 6 . In this study, fibrils of pGlu 3 -Aβ (3-40) of shorter length have been reported, which was not confirmed by our EM data. Although the pGlu modification on the N-terminus of truncated Aβ peptides significantly accelerates the fibrillation, the end product of this structure forming process shows an astonishing similarity to the well described structural features of all mature Aβ fibrils 15,16,23 . This suggests once more that the physiological effects of the pGlu peptides must be mediated by transient oligomers, which are very difficult to characterize. However, the N-terminus of the pGlu 3 -Aβ (3-40) fibrils showed dynamical alterations, that may have an effect of the stability of the intermediates as well as the fibrils as speculated before 6 . ThT fluorescence measurements. The fibrillation kinetics of pGlu 3 -Aβ (3-40) was followed by ThT fluorescence intensity measurements. Buffer conditions for fibrillation were the same as above with additional 20 μ M ThT in the incubation solution. Volumes of 150 μ l were pipetted into the wells of a 96-well plate, which was placed in a Tecan infinite M200 microplate reader (Tecan Group AG, Männedorf, Switzerland). The temperature was kept at 37 °C and a kinetic cycle was applied, such that a 2 s shaking time (2 mm shaking amplitude) followed by a 5 min waiting time was repeated four times with one additional 2 s shaking at the end and the subsequent fluorescence measurement. Fluorescence excitation was set to 440 nm and emission was measured at 482 nm. The fluorescence intensity was measured in increments of 30 min for an overall time period of 65 h. For comparison, also the kinetics of WT Aβ (1-40) fibrillation was recorded under the same conditions. Data was analyzed using procedures reported in the literature 36 . Electron microscopy. The fibril morphology was checked by electron microscopy (EM). Fibril solutions were diluted 1:1 with pure water and 1 μ l droplets of this solution were applied on formvar coated copper grids, allowed to dry for about 1 h and negatively stained with 1% uranyl acetate in pure water. Scanning transmission electron micrographs were recorded using Zeiss SIGMA, (Zeiss NTS, Oberkochen, Germany) equipped with a STEM detector and Atlas Software.

Sample preparation.
Solid-state MAS NMR spectroscopy. For NMR measurements, fibril solutions were ultracentrifuged at ~200,000× g for 4 h at 4 °C. The pellets were lyophilized, rehydrated to 50 wt% H 2 O, homogenized by several freeze-thaw cycles and finally transferred into 3.2 mm MAS rotors. All MAS NMR experiments were conducted on a Bruker 600 Avance III NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) at a resonance frequency of 600.1 MHz for 1 H, 150.9 MHz for 13 C, and 60.8 MHz for 15 N using a triple channel 3.2 mm MAS probe. Typical pulse lengths were 4 μ s for 1 H and 13 C and 5 μ s for 15 N. 1 H-13 C and 1 H-15 N CP contact time were 1 ms at a spin lock field of ~50 kHz. The relaxation delay was 2.5 s. 1 H dipolar decoupling during acquisition with a radio frequency amplitude of 65 kHz was applied using Spinal64. The MAS frequency was 11,777 Hz. 13 C chemical shifts were referenced externally relative to TMS.  Scientific RepoRts | 6:33531 | DOI: 10.1038/srep33531 13 C-13 C DARR NMR spectra and 13 C-15 N correlation spectra were acquired simultaneously using dual-acquisition 20 . In the same experiment, a two dimensional 13 C-13 C DARR NMR spectrum with a mixing time of 500 ms with 128 data points and four identical 15 N- 13 Cα correlation spectra with 32 data points in the indirect dimensions were measured. The 15 N-13 Cα spectra were processed using NMRPIPE software 37 .
To determine 1 H-13 C dipolar couplings, constant time DIPSHIFT experiments 38 were performed. For homonuclear decoupling during dipolar evolution a frequency switched Lee-Goldburg (FSLG) 39 with an effective radio frequency field of 80 kHz was used. The MAS frequency for DIPHSIFT experiments was 5 kHz. After Fourier transformation in the direct dimension the signal intensities of the dephasing curve for each resolved carbon was simulated and the determined coupling was divided by the known rigid limit values to obtain the order parameters 40,41 . The temperature for all NMR experiments was 30 °C. X-ray diffraction measurements. For X-ray diffraction measurements, fibril samples from the MAS rotors were placed on nylon loops (Hampton Research, Aliso Viejo, CA, USA) and mounted onto the goniometer head of a X-ray source (Rigaku copper rotating anode MM007 with 0.8 kW, Tokyo, Japan). The signals were recorded using an image plate detector (Rigaku, Tokyo, Japan) with an exposure time of 180 s at room temperature. Diffraction images were analyzed using ImageJ 42 .