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Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different Carolin Seuring, Joeri Verasdonck, Philippe Ringler, Riccardo Cadalbert, Henning Stahlberg, Anja Böckmann, Beat H Meier, and Roland Riek J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10624 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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The Journal of Physical Chemistry

Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different Carolin Seuring1, Joeri Verasdonck1, Philippe Ringler2, Riccardo Cadalbert1, Henning Stahlberg2, Anja 4* *

1*

Böckmann , Beat H. Meier , and Roland Riek 1

1,3*

Laboratory of Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093 Zürich, Switzerland

2

Center for Cellular Imaging and Nano Analytics (C-CINA), Biozentrum University of Basel, 4085 Basel, Switzerland

3

Structural Biology Laboratory, The Salk Institute, 10010 N Torrey Pines Road, 92037 La Jolla, CA,

USA 4

Molecular Microbiology and Structural Biochemistry, UMR 5086 CNRS, Université de Lyon 1, 7

passage du Vercors, 69367, Lyon, France *

Correspondence to A.B.: [email protected], B. M.: [email protected] or R. R.:

[email protected], +41 446326139 (tel) +41 446321021 (fax)

ACS Paragon Plus Environment


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Abstract Amyloid polymorphism of twisted and straight β-endorphin fibril was studied by negative-stain transmission electron microscopy, scanning transmission electron microscopy, and solid-state nuclear magnetic resonance spectroscopy. While fibrils assembled in the presence of salt form flat, striated ribbons, in the absence of salt they formed mainly twisted filaments. To get insights into their structural differences at the atomic level, 3D solid-state NMR spectra on both fibril types were acquired allowing the detection of the differences in chemical shifts of

13

C and

15

N atoms in both preparations. The

spectral fingerprints and therefore the chemical shifts are very similar for both fibril types. This indicates that the monomer structure and the molecular interfaces are almost the same but that these small differences do propagate to produce flat and twisted morphologies at the mesoscopic scale. This finding is in agreement with both experimental and theoretical considerations on the assembly of polymers (including amyloids) under different salt conditions that attribute the mesoscopic difference of flat versus twisted fibrils to electrostatic intermolecular repulsions.


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Introduction Amyloid deposits are found in post-mortem brains of patients with neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s Diseases and Huntington’s Disease1,2 One ubiquitous feature of disease-associated amyloids is amyloid polymorphism. Amyloid polymorphism describes the phenomenon that a single peptide/protein sequence can adopt structurally distinct conformations yielding different fibril morphologies at the mesoscopic scale (i.e. conformational polymorphism).3,4 While conformational polymorphs are believed to evoke different clinical characteristics and neuropathologies5,6 as for example in the case of prion strains7 or for α-synuclein8 the structural basis of the superstructures is yet less well understood.9-11 In this study we will focus on polymorphism exhibited through differences in fibril appearances at the mesoscopic level, which has been observed for numerous amyloid proteins and can either be co-present in a single fibril preparation or occur when fibrils are prepared under slightly different buffer conditions, e.g. by varying pH or salt concentrations.

12,13

Different fibril types in a fiber solution can be accessed by negative stain

transmission electron microscopy (TEM) and are often also distinct in assembly kinetics, binding properties to amyloid specific dyes such as thioflavin T, or/and may show differences in X-ray fiber diffraction as documented for example for glucagon,14 calcitonin,15 insulin,16 and amylin,17 prion protein (PrP),18 HET-s(218-289),19 α-synuclein,20 and Aβ21. Solid-state NMR chemical shifts are useful to access these differences at the atomic level because they are sensitive to small alterations in the local environment. In a polymorphic mixture, structural variety would either be visible as chemical-shift 22

differences, in the form of peak doubling for a set of resonances, or peak broadening.

In case of

different polymorphs, the spectral fingerprints generally differ considerably. This observation was demonstrated for different amyloids including IAPP,23 α-synuclein,20,24 Aβ(1-40),25,26 and tauprotein.

27,28

In these proteins, significant changes of the chemical shifts were observed for different

polymorphs, and in many cases the location of the β-sheets was different, indicating large structural changes at the molecular scale. 29-31

β-endorphin forms amyloid under several conditions.

Here, we have studied the twisted and flat-

striated fibril polymorphs of the functional hormone amyloid β-endorphin by negative-stain TEM and solid-state NMR formed at acidid pH. The β-endorphin amyloid is the dormant storage form of the βendorphin single hormone peptide

30,32

formed in cells of the pituitary gland. When soluble peptide

hormones are incubated for two weeks at a granule-relevant pH of 5.5 in the presence and absence of NaCl, straight and twisted forms of negative-stained β-endorphin fibrils were observed under the TEM microscope, respectively. In contrast to the examples listed above, the two types of fibrils show very similar solid-state NMR spectra with an identical positioning of the β-sheets. Considering the resolution of the spectra, which are comparable to spectra from microcrystalline protein ubiquitin or the functional highly-ordered HET-s(218-289) prion amyloid,33-37 we recon that the conformational differences between twisted and straight fibrils on the atomic scale must be small, but propagate from layer to layer within the fibril to strong differences that are observable at the mesoscopic scale.



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Experimental Methods Expression and purification of recombinant β-endorphin. β-endorphin was expressed in E. coli BL21* cells (Invitrogen) in a pET32a-vector as a thioredoxin fusion construct with an internal TEVprotease cleavage site. The entire method is described in Seuring et al..31 Briefly, the thioredoxin-βendorphin fusion protein was expressed with an N-terminal His-tag in M9-medium containing and of

15

NH4Cl

13

C-glucose as stable isotope sources. The lysate was purified under native and denaturing

conditions in buffer L (50 m M Tris pH 8.0, 300 mM NaCl, 20 mM imidazole, 10% v/v glycerol, 1mM DTT, 0.5 mg lysozyme/ml) in the presence and absence of 8 M Urea. After TEV-protease treatment (at a 1:40 molar ratio) at 20 °C for at least 4 hours, the sample was applied to a reverse phase C18 column (Supelco). Purified β-endorphin was obtained during a gradient of solvent A (0.1 % trifluoroacetic acid (TFA), 10 % acetonitrile (ACN)) and B (ACN 0.1 % TFA) at a flow rate of 2 ml/min at ~ 35 % of solvent B. Synthesis of natural abundance β-endorphin. Natural abundance β-endorphin was synthesized on an Applied Biosystems 433A automated batch peptide synthesizer. For the solid-phase peptide synthesis Fmoc-Glu(OtBu)-Wang-resin (Bachem: D-2330.0005) was used and commercial available Fmoc-amino acids. The commercial available labeled amino acids were modified with Fmocprotecting-group. The cleavage was made with the following cocktail: 9.4 ml TFA / 0.1 ml Triisopropylsilane (TIS) / 0.25 ml H2O / 0.25 ml Ethylenediamine tetraacetic acid. The cocktail was condensed away in vacuum. The crude product was washed with diethyl ether and the product was dried in vacuum. The obtained natural abundance β-endorphin was used for fibrillation. 30,31

Hormone fibril formation. Fibril formation was carried out based on the papers of Maji et al.

Briefly: purified recombinant or synthetic, lyophilized β-endorphin was dissolved at a concentration of 2 mg/ml in low-binding 1.5 ml Eppendorf tubes in either saline buffer (10 mM NH4Ac, 200 mM NaCl, 400 µM LMW heparin (5 kDa heparin from CalBioChem), 0.01 % azide, pH 5.5) or salt-free (5 % DMannitol, 0.01 % sodium azide, 400 µM LMW heparin (5 kDa heparin from CalBioChem), pH 5.530 and then incubated for two weeks at 37 °C under constant rotation using a rotating mixer (RT11, Torrey Pines Scientific). Solid-state NMR acquisition, data processing and spectra analysis. 2D and 3D spectra of uniformly

15

13

N, C-labeled -β-endorphin fibrils formed in saline buffer were recorded on a Bruker

Avance III operating at a static magnetic field of 20 Tesla. All spectra of fibrils incubated in the saltfree buffer were recorded on a Bruker Avance III spectrometer operating at a static magnetic field of 14.1 Tesla. The assignment procedure and acquisition details were described in detail previously.

38

Spectra were processed using Topspin 2.0 (Bruker Biospin), using a shifted cosine squared window function and zero-filling was done to the next power of two in all two or three dimensions. Automated polynomial baseline correction was applied in the direct dimension. Transmission electron microscopy (TEM) imaging. Samples of β-endorphin fibrils were diluted in buffer as required and adsorbed for 60 s to thin carbon films that span a thick fenestrated carbon layer covering 200-mesh/inch, gold-plated copper grids (STEM grids). The grids were then blotted, washed


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on 6 drops of quartz double distilled water and negatively stained with 2 % (w/v) uranyl acetate (UA). The grids where imaged with a CM 10 transmission electron microscope (Philips, Eindhoven, Netherlands) operating at 80 kV. Electron micrographs where recorded on a 2000 by 2000 pixel charge-coupled device camera (Veleta, Olympus soft imaging solutions GmbH, Münster, Germany) at a nominal magnification of 130,000 x yielding a final pixel size corresponding to 0.363 nm on the specimen scale. Scanning transmission electron microscopy (STEM) and mass-per- length (MPL) measurements. For mass measurements, freshly diluted aliquots of fibril solutions were adsorbed for 60 s to glow-discharged STEM grids (see above). The grids were then blotted, washed on 6 drops of quartz double distilled water to remove buffer salts, and freeze-dried at -80 °C and 5 x 108 Torr overnight in the microscope. Tobacco mosaic virus (TMV) par- ticles (kindly provided by R. Diaz Avalos, Institute of Molecular Biophysics, Florida State University) adsorbed to a separate grid and airdried served as mass standard. A Vacuum Generator (East Grinstead, U.K.) H5 STEM interfaced to a modular computer system (Tietz Video and Image Processing Systems) was used. Series of 512 x 512-pixel, dark-field images were recorded from the unstained sample at an acceleration voltage of 80 kV and a nominal magnification of 200,000 x. The recording dose range was between 320 and 780 electrons/nm2. Regions of the freeze-dried sample were also repeatedly scanned to determine the beam-induced mass-loss.39 The digital images were evaluated using the program package MASDET.

40

Fibril segments were selected in square boxes and tracked. The total scattering within an

integration box following their length was then calculated, and the scattering contribution of the supporting carbon film was subtracted. Division by the segment length gave the MPL. MPL values were corrected for beam-induced mass loss scaled to the MPL of tobacco mosaic virus, binned, displayed in histograms, and fitted by Gaussian curves. The overall experimental uncertainty was estimated from the corresponding standard error (SE = SD/√n) and the ~5 % uncertainty in the calibration of the instrument.



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Table 1. Comparison of saline and salt-free fibrils based on MPL measurements. “salt condition” : 10 mM NH4Ac, 200 mM NaCl and 400 µM heparin and 0.01% azide, at pH 5.5

“salt-free condition” : 5% D-Mannitol, 400 µM heparin and 0.01% azide at pH 5.5

kDa (STEM)

Width(STEM)

Filament Number *

Molecules/ nm**

kDa (STEM)

Width(STEM)

Filaments *

Molecules / nm

21

9

3

1.91

41.74

8.15

n.a.

n.a.

67.47

17.43

5.41

3.40

53.37

9.23

n.a

n.a.

128.74

32.78

10.18

3.45

61.02

10.51

n.a.

n.a.

* number of filaments were inferred indirectly, since they cannot be counted in STEM data. They were derived by deviding the “Width(STEM)” by the width obtained from TEM images. To indicate, that the filament number were obtained indirectly the values are written in italics. ** The molecules per nm were calculated using the number of filaments (column 3), which were determined only indirectly as stated in *. To indicate, that the molecules per nm were obtained only indirectly the values are written in italics.


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Results and Discussion β-endorphin fibrils can be classified in two polymorphs by TEM: striated ribbons and twisted fibrils For structural studies β-endorphin fibrils were prepared under two different buffer conditions (i.e. either 10 mM NH4Ac and 200 mM NaCl, or 5% mannitol30) in the presence of 400 µM heparin at pH 5.5 31

(Table 1). In the following, we will simply refer to the two buffer conditions as saline buffer condition

30

and salt-free

containing salt (saline) and no salt (salt-free), respectively.

When viewed by negative-stain transmission electron microscopy, in a saline environment βendorphin primarily fibrillizes into flat and striated ribbons (Figure 1A). These saline fibers generally consist of more than three protofilaments, which associate laterally with equidistant spacings. The number of protofilaments in a fiber can be deduced from the TEM image of the fiber by summing all pixel intensities of every column in the image to a single value per column. As an example, in Figure 1A the column sums of 18 nm, 24 nm, and 30 nm thick fibrils are plotted below their enlarged TEM micrographs exemplifying their corresponding 1D intensity profile. By counting the number of peaks in the 1D plot, the 18 nm, 24 nm, and 30 nm fibers consist of three, four, and five laterally packed filaments. The central filament in the 18 nm fiber gives a double intensity in the 1D profile, indicative for a stacking of the protofilaments into three dimensions. Confidence for this 3D packing can be retrieved more clearly from scanning transmission electron microscopy (STEM) on the same but frozen and unstained fibril preparation (Figure 2). This quantitative technique is sensitive to the number of electrons absorbed: the more filaments are present in the electron beam, the more electrons are absorbed and the brighter the fibrils appear. By simply applying a color gradient to the different white tones, the 3D layering becomes clear to the eye (Figure 2). To derive the full width at half maximum (FWHM) of saline protofilaments, we identified 18 protofilaments without 3D packing in TEM images using their 1D intensity profiles, which were fitted to a Gaussian function with a unique amplitude (A), a center position (b) and standard deviation (σ) per fiber. Based on the computed standard deviations of the Gaussians, we derived the FWHM of the fiber . All fits are shown in Figure S1A and the corresponding values are given in Table S1. The mean FWHM for salt fibers obtained is equal to 8.9 ± 0.7 pixels, or 3.2 ± 0.2 nm. Contrarily, in a salt-free environment, micrometer long fibrils very abundantly twist around their fibril axis as observed by TEM (Figure 1B). The twists are very regular, when two filaments associate laterally. If more than two protofilaments associate, twists become less pronounced and less regular. A close-up is given in Figure 1B (right) showing that the mean FWHM for salt-free fibers is difficult to retrieve due to the following reasons: (1) their 1D profiles merge indicative of a closer filament packing, (2) fibers are twisted, and (3) single fibers are rare and give low signal/noise ratios. Taking this into account, the mean width of the smallest filament prepared without salt is calculated to be 7.8 ± 0.4 pixels, or 2.8 ± 0.2 nm. All values derived from the Gaussians shown in Figure S1B are listed in order from left to right in Table S1.



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Mass-per-length measurements on fibrils prepared in saline and salt-free buffer reveal different protofilament packing As another structural comparison, the mass-per-length (MPL) values from the STEM measurements introduced above were obtained on frozen-hydrated samples of saline striated ribbons and salt-free twisting fibers. MPL measurements were obtained of individual fibrils (Figure 3) and accompanied by corresponding measurements of the fibril widths (Figure S2). A total of 552 measurements were drawn from STEM images of straight β-endorphin fibrils grown in saline buffer. The histogram was fitted to four Gaussians, which retrieve four maxima at MPL values of 22.2 ± 0.5, 39.2 ± 0.7, 53.6 ± 0.5, and 72.8 ± 1.3 kDa/nm. (Please note, for the fit, we needed to take the surrounding very small peaks at ~ 10.9 kDa/nm and ~ 87.5 kDa/nm into account, but we were not able to retrieve their exact maxima.) The difference of the MPL values between each neighboring values are with 16.6 ± 2 kDa/nm similar supporting the finding that a variable number of the same protofilaments assemble into these ribbons. All mass-per-length values are related to the number of molecules packed along the fibril axis over one nanometer and can be obtained from:

, where molecules is number of individual βendorphin peptides, MW(STEM) is the experimentally obtained molecular weight in kDa, and MW(βendorphin) is the molecular weight of β-endorphin (i.e. 3.47 kDa) used. In STEM images (Figure S2) the smallest fibers are ~ 9 nm and the histogram reveals the smallest MPL value of 22.2 kDa/nm. We further assume that a 9 nm fiber consists of three protofilaments based on the averaged FWHM of ~ 3.22 nm, which was obtained from Gaussian fits (Figure S1, Table S1). Following this rational, the mass-per-length variation of 16.6 ± 2 kDa/nm corresponds to two molecules per nanometer and correspondingly one molecule per cross-β-sheet layer30 (by using the inter β-strand distance of 0.48 nm). On twisted β-endorphin fibrils grown in salt-free buffer 58 STEM MPL values and widths measurements were acquired (Figure S2B). In total, three classes of fibril widths accordant with a different number of protofilaments were retrieved. We measured widths of 8.2 ± 0.4, 9.2 ± 0.4, and 10.5 ± 0.4 nm corresponding to MPL values of 41.7 ± 4, 53.4 ± 4, and 61.0 ± 4 kDa/nm. The 41.7 kDa/nm and 53.4 kDa/nm fibers have a counterpart in the saline preparation, while the 61 kDa/nm value was not fitted by the Gaussians due to very few measurements of this value (Figure 3). Nonetheless, we have observed a filament of 67.5 ± 2.3 kDa/nm also in saline preparations (Figure S2). It seems obvious to explain the increase in weight with the addition of one protofilament (i.e. 7.2 kDa/nm). Interestingly the widths values of the circa 42, 54 and 61 kDa fibrils shows the same difference between each class of ~1.2 nm. In contrast to the TEM measurements with a FWHM of ~2.8 nm (see above), this smaller diameter could be the second dimension of the β-endorphin fibril. Along this line, the increase in weight between the ~42 and ~61 kDa fibrils can be explained by the difference of two protofilaments, whereof each has a MPL of ~ 9 kDa/nm. This value corroborates with the MPL of one molecule per cross-β-sheet layer as observed in saline fibers.


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Solid-state NMR spectra of striated and twisted fibrils are very similar To further investigate the polymorphic nature of β-endorphin fibrils prepared in salt-free and saline buffer at atomic resolution, magic-angle spinning (MAS) solid-state NMR spectra were acquired on 15

N,13C-labeled material (Figure 4). Solid-state NMR spectra of saline and salt-free fibers are well 13

C-resonances in salt-free

resolved and indicative for well-ordered fibrils. The typical line-width of the

preparations are around ~120 Hz (~0.5 ppm) at a static magnetic fields of 850 MHz (20.0 Tesla) and thus comparable to spectra of saline preparations, which we have reported previously.31 In Figure 4, we overlaid the aliphatic chemical shifts of 13

C-labelled striated ribbons (blue). Their

13

15

13

N, C-labelled twisted fibers (red) and

15

N,

13

C- C correlation solid-state NMR 2D DARR spectra

appear to be almost identical (Figure 4), as do their

15

N-13C 2D NCA spectra (Figure S3). In order to

quantify chemical shift-differences, 3D experiments were used to obtain the chemical-shift assignment for saline31 and salt-free fibrils. The assignment of 94 % of the

13

C- and

15

N-atoms in

15

N, 13C-labeled

β-endorphin amyloid prepared in saline buffer amyloid fibrils was previously reported (BMRB 31

accession number 26715).

In this work, the sequential assignment of β-endorphin fibrils grown in

salt-free buffer was obtained based on a combination of two 3D experiments (NCACX and NCOCA)

41,42

plus two 2D experiments (NCO and NCA). Figure S5 shows a side-by-side comparison

of a stretch of the sequential walk of the four-amino-acid-residues segment 17 to 14 of β-endorphin prepared in salt-free buffer and saline buffer. The assignment of salt-free fibrils is deposited under the BMRB accession number 26900 and is complete to 79% of the The difference of the secondary chemical shifts between the

13

13

C- and

Cα and

15

N-atoms.

13

Cβ resonances43,44 is shown

in Figure 5 for the two polymorphs. From these values, the secondary structure of the peptide can be predicted.

45

Three or more negative values in a row are indicative of a β-strand. As a measure of the

conformational differences in saline and salt-free samples, the secondary chemical shifts of the two polymorphs are compared in Figure 5A. The error bars are derived from the standard deviation of the peak position between different peaks with the same assignment as given by CCPN. The chemicalshift differences between the polymorphs are shown in Figure 5B. The size of the chemical-shift differences is comparable to the error in the peak position. The secondary chemical shift analysis is consistent with equal β-strand positions along the peptide sequence in both polymorphs. To evaluate the sample and measurement-specific chemical shift accuracy, we have used the atomspecific single standard deviation of peak positions between different spectra of the same polymorph as a measure for the measurement error of the assignment. For the standard deviation, chemical shifts of the same polymorph were compared (i) between different spectra and (ii) different sample preparations. In Figure 6A, we depicted two peaks, Phe 4 (13Cα/13Cβ) (red) and Phe 18 (13Cα/13Cβ) (blue) in four spectra acquired on exactly the same sample but in different measuring sessions and with different pulse schemes. Extracts of a 20 ms DARR, 8 ms PAR, 400 ms DARR, and a 500 us CHHC spectra are shown. The red and blue crosshairs are at the same peak position in all four panels. It is obvious that the peak centers of Phe 4 and Phe 18 exhibit small differences with a maximum deviation from their corresponding center point by roughly ±0.15 ppm from panel to panel. This difference is used as a rough measure for the precision of our measurements. It is similar to the



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difference observed between the shift values of the two polymorphs. In addition to these small chemical-shift variations between different spectra, we also looked at the difference between spectra 13

13

of two different fibril batches. As shown in Figure 6B for Phe 4 ( Cα/ Cβ) (red) and Phe 18 (13Cα/13Cβ) (blue) the maximum peak position varies between batches to a similar extend as it varies between spectra types. This analysis shows that the small chemical-shift differences between the two polymorphs mentioned above (Figure 5) are of the same order as the estimated accuracy of our measurement. For only five residues (i.e. Phe 4, Lys 9, Gln 11, Lys 19, and Lys 24) the difference between the secondary chemical shifts is slightly larger than one standard deviation. Next, we have compared the chemical shift of every 13C atom assigned in both samples. The absolute values for the cumulative

13

C chemical shift differences per residue |δ(13C Saline Buffer) - δ(13C Salt-free Buffer)| in

ppm is plotted in Figure S6. The summed differences in ppm for most amino acids are around 0.4 ppm or smaller, even for residues with long side-chains. More noticeable differences are seen for the aromatic residues Phe 4, Phe 18 and Tyr 27, as well as for Lys 28. The difference in

15

N chemical

shifts is shown in Figure S4. Again only small differences were found with a maximum of 0.6 ppm for Lys 28.

Discussion Twisted and straight β-endorphin fibrils appear to have very different morphologies when observed via negative stain and scanning TEM (Figure 1). In contrast to the straight fibrils, the thinnest twisted fibrils rotate every ~150 nm by 360° (Figure 1). In addition, the number of protofilaments, their widths and their mass per length values are considerably different (Figures 1, Figure S1, Figure 3). At first it appears thus surprising that no significant differences in molecular conformation are found between the two polymorphs using the solid-state NMR spectra (Figure 4, Figure 6, Figure S5) indicating that minor structural differences at the molecular level may give rise to large effects at the mesoscopic scale. This can be rationalized by calculating the twist rate per molecule which is in the range of 0 ° for the straight fibrils and 0.8 ° for the twisted fibril by taking into account the MPL measurements of the fibers (MPL/nm ~ 2 molecules), which suggest that every layer (0.47 nm) of the cross-β-sheet is formed by one β-endorphin molecule. While a twist of 0.8 ° per molecule changes the local structure including the intermolecular hydrogen bond length only very slightly and is thus apparently not observed in the solid-state NMR spectra, the effect propagates surprisingly strongly resulting in entirely differently appearing fibrils under the electron microscope. The nature of the observed difference at the mesoscopic level must be attributed to the salt concentration. Indeed, also for other protein amyloids, salt-dependent twist rates from flat, to different degrees of twists up to round (closed) ribbons have been observed and discussed in details.46,47 These findings have been attributed to intermolecular electrostatic repulsion within the amyloid fibrils that cause twisting, while salt is capable of alleviating the twisting by interfering with the electrostatic repulsion. Interestingly, such observations appear to be a general phenomenon for polymers as both documented and theoretically described.

48,49

Showing only little if at all structural differences at the

atomic level of both twisted and flat amyloids, the presented studies are further supportive of this concept.


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Also the observed anti-correlation between the twisting rate of the amyloid and the lateral association of protofilaments (Figure 1) is well documented and understood in the amyloid field.50 It has been shown that twisted amyloid fibrils increase their periodicity linearly with the number of protofilaments

51

up to a critical number of protofilaments (i.e. at a critical width-to-thickness ratio) at which a topological transition from twisted to helical filaments is observed and it can be explained by minimization of bending versus torsional energy avoiding thereby the outer filaments to run a longer way around the fibril axis in twisted amyloids.52,53 The observation and interpretation of small salt-induced structural changes at the molecular level that show significant differences at the mesoscopic level is thus in line with the suggestion that functional amyloids as the hormone of interest here do not polymorphisms because they have been evolutionary optimized, while polymorphisms of disease-associated amyloids are wide spread and well documented.3,4 Interestingly, the latter are observed on both the mesoscopic level using for example EM, as well as at the atomic level showing distinct solid-state NMR spectra.

3,4

This indicates that these

polymorphs - some of which are induced by different salt concentrations - are significantly different at the atomic scale with distinct three dimensional fibrillar core structures that propagate to distinguishable fibril types at the mesoscopic scale.

3,4,20,24

This contrasts to the presented polymorphs

of β-endorphin fibrils, that, based on the solid-state NMR spectra have the same amyloid core structures as expected from an evolutionary optimization point of view. Rather than biologically relevant, this finding may therefore be regarded a prime example of one of the many amazing activities of the repetitive structural nature of amyloids: the propagation of a miniscule structural difference into a strong one through propagation over several orders of scale from the sub-Ångstrom scale to the micrometer scale. Interestingly, it appears that this difference can be manipulated by the environmental conditions such as the salt content of the buffer system. Conclusion According to the hypothesis by Maji et al.,30 hormones stored in secretory granules of the pituitary gland form functional amyloid fibrils. In order to get a detailed structural characterization of this class of functional human amyloids, the human β-endorphin amyloid was studied in detail exploiting solidstate NMR spectroscopy and TEM analyses. Upon incubation under two physiological relevant buffer conditions, recombinant stable-isotope labeled β-endorphin fibrils were grown. The solid-state NMR spectra obtained thereof enabled an almost complete sequential assignment of both polymorphs, for which the chemical shifts are very similar, in fact not larger than the experimental precision. Furthermore, both β-endorphin fibrils share the same line widths in solid-state NMR spectra. The local order of both fibrils has thus to be very similar indicating that the propagation of minor local structural difference may lead to different overall fibril morphologies at the mesoscopic scale.



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Acknowledgement This work was supported by the Swiss National Foundation and an ETH-internal grant. We would like to thank Julia Gath for support with NMR spectra acquisition and corrections with this paper. Many thanks to Marielle Wälti for fiber preparation for additional STEM measurements and Joseph Wall for acquiring STEM data at Brookhaven National Laboratory. Rick Millane and David Wojtas from the University of Canterbury for discussing the Gaussian fittings. Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: . Figure of negative-stain TEM images of saline and salt-free fibrils and their corresponding intensity profiles; figure of the STEM analysis of saline and salt-free fibrils; the overlay of of salt-free and saline fibrils; bar plot showing the differences in amide

15

N-13C NCA spectra

15

N chemical shift between the

saline and salt-free samples; side-by-side comparison of the sequential back-bone assignments for saline and salt-free fibers; bar plot of the averaged absolute values between the mean chemical shift differences computed from both fibril types; and a negative-stain TEM image of highly bundling fibrils in saline buffer.


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References

(1) Eisenberg, D.; Jucker, M., The Amyloid State of Proteins in Human Diseases. Cell 2012, 148, 1188-1203. (2) Chiti, F.; Dobson, C. M., Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333-366. (3) Tycko, R., Physical and Structural Basis for Polymorphism in Amyloid Fibrils. Protein Sci. 2014, 23, 1528-1539. (4) Tycko, R., Amyloid Polymorphism: Structural Basis and Neurobiological Relevance. Neuron 2015, 86, 632-645. (5) Riek, R.; Eisenberg, D. S., The Activities of Amyloids from a Structural Perspective. Nature 2016, 539, 227-235. (6) Stein, K. C.; True, H. L., Prion Strains and Amyloid Polymorphism Influence Phenotypic Variation. PLoS Pathog. 2014, 10. (7) Toyama, B. H.; Kelly, M. J.; Gross, J. D.; Weissman, J. S., The Structural Basis of Yeast Prion Strain Variants. Nature 2007, 449, 233-237. (8) Peelaerts, W.; Bousset, L.; Van der Perren, A.; Moskalyuk, A.; Pulizzi, R.; Giugliano, M.; Van den Haute, C.; Melki, R.; Baekelandt, V., Alpha-Synuclein Strains Cause Distinct Synucleinopathies after Local and Systemic Administration. Nature 2015, 522, 340-344. (9) Bieschke, J.; Herbst, M.; Wiglenda, T.; Friedrich, R. P.; Boeddrich, A.; Schiele, F.; Kleckers, D.; Lopez del Amo, J. M.; Gruning, B. A.; Wang, Q., et al., Small-Molecule Conversion of Toxic Oligomers to Nontoxic Beta-Sheet-Rich Amyloid Fibrils. Nat. Chem. Biol. 2012, 8, 93-101. (10) Vetri, V.; Fodera, V., The Route to Protein Aggregate Superstructures: Particulates and Amyloid-Like Spherulites. FEBS Lett. 2015, 589, 2448-2463. (11) Krebs, M. R.; Macphee, C. E.; Miller, A. F.; Dunlop, I. E.; Dobson, C. M.; Donald, A. M., The Formation of Spherulites by Amyloid Fibrils of Bovine Insulin. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14420-14424. (12) Verasdonck, J.; Bousset, L.; Gath, J.; Melki, R.; Bockmann, A.; Meier, B. H., Further Exploration of the Conformational Space of Alpha-Synuclein Fibrils: Solid-State Nmr Assignment of a High-Ph Polymorph. Biomol. NMR Assignments 2016, 10, 5-12. (13) Walti, M. A.; Ravotti, F.; Arai, H.; Glabe, C. G.; Wall, J. S.; Bockmann, A.; Guntert, P.; Meier, B. H.; Riek, R., Atomic-Resolution Structure of a Disease-Relevant Abeta(1-42) Amyloid Fibril. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E4976-4984. (14) Andersen, C. B.; Hicks, M. R.; Vetri, V.; Vandahl, B.; Rahbek-Nielsen, H.; Thogersen, H.; Thogersen, I. B.; Enghild, J. J.; Serpell, L. C.; Rischel, C., et al., Glucagon Fibril Polymorphism Reflects Differences in Protofilament Backbone Structure. J. Mol. Biol. 2010, 397, 932-946. (15) Bauer, H. H.; Aebi, U.; Haner, M.; Hermann, R.; Muller, M.; Merkle, H. P., Architecture and Polymorphism of Fibrillar Supramolecular Assemblies Produced by in Vitro Aggregation of Human Calcitonin. J. Struct. Biol. 1995, 115, 1-15. (16) Jimenez, J. L.; Nettleton, E. J.; Bouchard, M.; Robinson, C. V.; Dobson, C. M.; Saibil, H. R., The Protofilament Structure of Insulin Amyloid Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 9196-9201. (17) Goldsbury, C. S.; Cooper, G. J. S.; Goldie, K. N.; Muller, S. A.; Saafi, E. L.; Gruijters, W. T. M.; Misur, M. P., Polymorphic Fibrillar Assembly of Human Amylin. J. Struct. Biol. 1997, 119, 17-27.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(18) Cobb, N. J.; Apostol, M. I.; Chen, S. G.; Smirnovas, V.; Surewicz, W. K., Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region. J. Biol. Chem. 2014, 289, 2643-2650. (19) Wan, W.; Wille, H.; Stohr, J.; Baxa, U.; Prusiner, S. B.; Stubbs, G., Degradation of Fungal Prion Het-S(218-289) Induces Formation of a Generic Amyloid Fold. Biophys. J. 2012, 102, 2339-2344. (20) Bousset, L.; Pieri, L.; Ruiz-Arlandis, G.; Gath, J.; Jensen, P. H.; Habenstein, B.; Madiona, K.; Olieric, V.; Bockmann, A.; Meier, B. H., et al., Structural and Functional Characterization of Two Alpha-Synuclein Strains. Nat. Commun. 2013, 4, 2575. (21) Colletier, J. P.; Laganowsky, A.; Landau, M.; Zhao, M. L.; Soriaga, A. B.; Goldschmidt, L.; Flot, D.; Cascio, D.; Sawaya, M. R.; Eisenberg, D., Molecular Basis for Amyloid-Beta Polymorphism. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16938-16943. (22) Meier, B. H.; Bockmann, A., The Structure of Fibrils from 'Misfolded' Proteins. Curr. Opin. Struct. Biol. 2015, 30, 43-49. (23) Luca, S.; Yau, W. M.; Leapman, R.; Tycko, R., Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State Nmr. Biochemistry 2007, 46, 13505-13522. (24) Gath, J.; Bousset, L.; Habenstein, B.; Melki, R.; Bockmann, A.; Meier, B. H., Unlike Twins: An Nmr Comparison of Two Alpha-Synuclein Polymorphs Featuring Different Toxicity. PLoS One 2014, 9, e90659. (25) Paravastu, A. K.; Leapman, R. D.; Yau, W. M.; Tycko, R., Molecular Structural Basis for Polymorphism in Alzheimer's Beta-Amyloid Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18349-18354. (26) Petkova, A. T.; Leapman, R. D.; Guo, Z.; Yau, W. M.; Mattson, M. P.; Tycko, R., SelfPropagating, Molecular-Level Polymorphism in Alzheimer's Beta-Amyloid Fibrils. Science 2005, 307, 262-265. (27) Andronesi, O. C.; von Bergen, M.; Biernat, J.; Seidel, K.; Griesinger, C.; Mandelkow, E.; Baldus, M., Characterization of Alzheimer's-Like Paired Helical Filaments from the Core Domain of Tau Protein Using Solid-State Nmr Spectroscopy. J. Am. Chem. Soc. 2008, 130, 5922-5928. (28) Daebel, V.; Chinnathambi, S.; Biernat, J.; Schwalbe, M.; Habenstein, B.; Loquet, A.; Akoury, E.; Tepper, K.; Muller, H.; Baldus, M., et al., Beta-Sheet Core of Tau Paired Helical Filaments Revealed by Solid-State Nmr. J. Am. Chem. Soc. 2012, 134, 13982-13989. (29) Nespovitaya, N.; Gath, J.; Barylyuk, K.; Seuring, C.; Meier, B. H.; Riek, R., Dynamic Assembly and Disassembly of Functional Beta-Endorphin Amyloid Fibrils. J Am Chem Soc 2016, 138, 846-856. (30) Maji, S. K.; Perrin, M. H.; Sawaya, M. R.; Jessberger, S.; Vadodaria, K.; Rissman, R. A.; Singru, P. S.; Nilsson, K. P.; Simon, R.; Schubert, D., et al., Functional Amyloids as Natural Storage of Peptide Hormones in Pituitary Secretory Granules. Science 2009, 325, 328-332. (31) Seuring, C.; Gath, J.; Verasdonck, J.; Cadalbert, R.; Rivier, J.; Bockmann, A.; Meier, B. H.; Riek, R., Solid-State Nmr Sequential Assignment of the Beta-Endorphin Peptide in Its Amyloid Form. Biomol. NMR Assignments 2016. (32) Dannies, P. S., Protein Hormone Storage in Secretory Granules: Mechanisms for Concentration and Sorting. Endocr. Rev. 1999, 20, 3-21. (33) Igumenova, T. I.; McDermott, A. E.; Zilm, K. W.; Martin, R. W.; Paulson, E. K.; Wand, A. J., Assignments of Carbon Nmr Resonances for Microcrystalline Ubiquitin. J. Am. Chem. Soc. 2004, 126, 6720-6727. (34) Castellani, F.; van Rossum, B. J.; Diehl, A.; Rehbein, K.; Oschkinat, H., Determination of Solid-State Nmr Structures of Proteins by Means of Three-Dimensional ACS Paragon Plus Environment

Page 14 of 23

Page 15 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15n-13c-13c Dipolar Correlation Spectroscopy and Chemical Shift Analysis. Biochemistry 2003, 42, 11476-11483. (35) Wasmer, C.; Lange, A.; Van Melckebeke, H.; Siemer, A. B.; Riek, R.; Meier, B. H., Amyloid Fibrils of the Het-S(218-289) Prion Form a Beta Solenoid with a Triangular Hydrophobic Core. Science 2008, 319, 1523-1526. (36) Van Melckebeke, H.; Wasmer, C.; Lange, A.; Ab, E.; Loquet, A.; Bockmann, A.; Meier, B. H., Atomic-Resolution Three-Dimensional Structure of Het-S(218-289) Amyloid Fibrils by Solid-State Nmr Spectroscopy. J. Am. Chem. Soc. 2010, 132, 13765-13775. (37) Siemer, A. B.; Ritter, C.; Ernst, M.; Riek, R.; Meier, B. H., High-Resolution SolidState Nmr Spectroscopy of the Prion Protein Het-S in Its Amyloid Conformation. Angew. Chem. Int. Ed. Engl. 2005, 44, 2441-2444. (38) Seuring, C.; Greenwald, J.; Wasmer, C.; Wepf, R.; Saupe, S. J.; Meier, B. H.; Riek, R., The Mechanism of Toxicity in Het-S/Het-S Prion Incompatibility. PLoS Biol 2012, 10, e1001451. (39) Muller, S. A.; Goldie, K. N.; Burki, R.; Haring, R.; Engel, A., Factors Influencing the Precision of Quantitative Scanning-Transmission Electron-Microscopy. Ultramicroscopy 1992, 46, 317-334. (40) Krzyzanek, V.; Muller, S. A.; Engel, A.; Reichelt, R., Masdet-a Fast and UserFriendly Multiplatform Software for Mass Determination by Dark-Field Electron Microscopy. J. Struct. Biol. 2009, 165, 78-87. (41) Pauli, J.; Baldus, M.; van Rossum, B.; de Groot, H.; Oschkinat, H., Backbone and Side-Chain 13c and 15n Signal Assignments of the Alpha-Spectrin Sh3 Domain by Magic Angle Spinning Solid-State Nmr at 17.6 Tesla. Chembiochem 2001, 2, 272-281. (42) Schuetz, A.; Wasmer, C.; Habenstein, B.; Verel, R.; Greenwald, J.; Riek, R.; Bockmann, A.; Meier, B. H., Protocols for the Sequential Solid-State Nmr Spectroscopic Assignment of a Uniformly Labeled 25 Kda Protein: Het-S(1-227). Chembiochem 2010, 11, 1543-1551. (43) Luginbuhl, P.; Szyperski, T.; Wuthrich, K., Statistical Basis for the Use of C-13Alpha Chemical-Shifts in Protein-Structure Determination. J. Magn. Reson. B 1995, 109, 229-233. (44) Wang, Y.; Jardetzky, O., Probability-Based Protein Secondary Structure Identification Using Combined Nmr Chemical-Shift Data. Protein Sci. 2002, 11, 852-861. (45) Wishart, D. S.; Sykes, B. D., The C-13 Chemical-Shift Index - a Simple Method for the Identification of Protein Secondary Structure Using C-13 Chemical-Shift Data. J. Biomol. NMR 1994, 4, 171-180. (46) Adamcik, J.; Mezzenga, R., Adjustable Twisting Periodic Pitch of Amyloid Fibrils. Soft Matter 2011, 7, 5437-5443. (47) Assenza, S.; Adamcik, J.; Mezzenga, R.; De Los Rios, P., Universal Behavior in the Mesoscale Properties of Amyloid Fibrils. Phys. Rev. Lett. 2014, 113. (48) Sawa, Y.; Ye, F.; Urayama, K.; Takigawa, T.; Gimenez-Pinto, V.; Selinger, R. L.; Selinger, J. V., Shape Selection of Twist-Nematic-Elastomer Ribbons. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6364-6368. (49) Oda, R.; Huc, I.; Schmutz, M.; Candau, S. J.; MacKintosh, F. C., Tuning Bilayer Twist Using Chiral Counterions. Nature 1999, 399, 566-569. (50) Adamcik, J.; Mezzenga, R., Proteins Fibrils from a Polymer Physics Perspective. Macromolecules 2012, 45, 1137-1150. (51) Adamcik, J.; Jung, J. M.; Flakowski, J.; De Los Rios, P.; Dietler, G.; Mezzenga, R., Understanding Amyloid Aggregation by Statistical Analysis of Atomic Force Microscopy Images. Nat. Nanotechnol. 2010, 5, 423-428.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(52) Adamcik, J.; Castelletto, V.; Bolisetty, S.; Hamley, I. W.; Mezzenga, R., Direct Observation of Time-Resolved Polymorphic States in the Self-Assembly of End-Capped Heptapeptides. Angew Chem Int Edit 2011, 50, 5495-5498. (53) Lara, C.; Adamcik, J.; Jordens, S.; Mezzenga, R., General Self-Assembly Mechanism Converting Hydrolyzed Globular Proteins into Giant Multistranded Amyloid Ribbons. Biomacromolecules 2011, 12, 1868-1875.


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Figure 1



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Figure 2.


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Figure 3.



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Figure 4.


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Figure 5.



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Figure 6.


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