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Electrostatic Interactions at N- and C-termini Determine Fibril Polymorphism in Serum Amyloid A Fragments Justine M. Jannone, James I. Grigg, Lauren M. Aguirre, and Eric M. Jones J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b07672 • Publication Date (Web): 15 Sep 2016 Downloaded from http://pubs.acs.org on September 16, 2016

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The Journal of Physical Chemistry

Electrostatic Interactions at N- and C-Termini Determine Fibril Polymorphism in Serum Amyloid A Fragments

Justine M. Jannone, James I. Grigg, Lauren M. Aguirre, and Eric M. Jones* Department of Chemistry and Biochemistry, California Polytechnic State University San Luis Obispo, CA 93407 USA

*

Corresponding author: Department of Chemistry and Biochemistry, 1 Grand Ave., San Luis

Obispo, CA 93407; ph (805) 756-2425; fx (805) 756-5500, email [email protected]

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ABSTRACT: Amyloid polymorphism presents a challenge to physical theories of amyloid formation and stability. The amyloidogenic protein serum amyloid A (SAA) exhibits complex and unexplained structural polymorphism in its N-terminal fragments: the N-terminal 11-residue peptide (SAA1-11) forms left-handed helical fibrils, while extension by one residue (SAA1-12) produces a rare right-handed amyloid.

In this study, we use a combination of vibrational

spectroscopy and ultramicroscopy to examine fibrils of these peptides and their terminally acetylated and amidated variants, in an effort to uncover the physical basis for this effect. Raman spectroscopy and atomic force microscopy provide evidence that SAA1-12 forms a βhelical fibril architecture, while SAA1-11 forms more typical stacked β-sheets. Importantly, Nterminal acetylation blocks fibril formation by SAA1-12 with no effect on SAA1-11, while Cterminal amidation has nearly the opposite effect.

Together, these data suggest distinct

electrostatic interactions at the N- and C-termini stabilize the two fibril structures; we propose model fibril structures in which C-terminal extension changes the favored intermolecular interaction between peptide monomers from an Arg1 – C-terminus charge pair to an N-terminus – C-terminus charge pair.

This model suggests a general mechanism for charge-mediated

amyloid polymorphism, and may inform strategies for design of peptide-based nanomaterials stabilized by engineered intermolecular contacts.


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INTRODUCTION Recent decades have seen a surge of interest in the structure and properties of amyloid proteins and peptides. Identified originally for their association with debilitating diseases,1–3 increased understanding of the physics and chemistry of amyloid formation has led interest in these protein aggregates for practical applications in bionanotechnology and biomaterials science.4–6 Despite numerous advances, a generalized understanding of the physical basis of amyloid formation remains elusive. One of the confounding problems in amyloid chemistry is the existence of polymorphism, or the ability of proteins of identical or very similar sequence to form multiple amyloid structures.7–11

This phenomenon can account for differing disease phenotypes in

amyloidoses,12–16 and in particular is responsible for the existence of “strains” and species barriers in prion disorders.14,17–21 With the discovery of varying amyloid polymorphs in a wide variety of amyloid proteins, it is becoming clear that polymorphism is a general and possibly universal feature of amyloids, resulting from (mis)folding on a complex energy landscape.7,10,21 Serum amyloid A (SAA) is a component of high-density lipoproteins overexpressed in response to proinflammatory cytokines,22 resulting in AA amyloidosis.23

Full-length SAA1.1

terminalamino acids (shared among all human SAA isoforms24,30) (the pathogenic SAA variant) is a mostly α-helical protein (Figure 1) that assembles into a homohexamer,24 but the highly hydrophobic N-terminal helix is particularly crucial to amyloidogenesis by the protein25–27 and forms amyloid fibrils in isolation,24 though the C-terminus also plays a modulating role in fibrillogenesis.28 N-terminal truncations of SAA1.1, in which the first one or two amino acids are cleaved, have furthermore been specifically associated with diabetes-associated AA amyloidosis.29 These studies point to a crucial role of N- in determining the amyloidogenicity and pathogenicity of SAA1.1. Page 3 of 37 ACS Paragon Plus Environment


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Figure 1: Top, structure of the SAA monomer unit (PDB ID 4ip9), with residues 1-12 highlighted in yellow. Sidechains forming contacts with the dimer and trimer interfaces are colored magenta and salmon-pink, respectively. Bottom, sequences of the peptides used in this study. Figure generated using PyMOL 68. In two studies of N-terminal SAA peptides, Rubin et al.31,32 found that short peptide fragments of SAA display a striking example of polymorphism: amyloids formed by the first 11 residues of SAA (SAA1-11) form fibrils with a classic left-handed helical morphology, while extension of this peptide by one residue, forming SAA1-12, resulted in fibrils with a rare righthanded helical morphology. This morphological change was not sensitive to the identity of the amino acid in the twelfth position, and persisted in the presence of an N-terminal truncation to SAA2-12,31 indicating that neither length nor sequence per se is responsible for this structural Page 4 of 37 ACS Paragon Plus Environment

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change. Polymorphism has also been observed in full-length SAA1.1 fibrils formed under different solution conditions.26 In this study, we attempt to establish the origin of this dramatic case of amyloid polymorphism. We show, using Raman and infrared spectroscopy, that the right-handed SAA112 amyloids contain considerable disordered and α-helical conformation, consistent with a βhelical architecture, while SAA1-11 is almost entirely β-sheet. Fibril cross-sections, measured using atomic force microscopy (AFM), indicate SAA1-11 can form unusually thin amyloids (comparable to a single protofilament in some cases) whereas SAA1-12 exclusively forms thicker fibrils. We also find that N-terminal acetylation abolishes fibril formation by SAA1-12 but has no effect on SAA1-11, while C-terminal amidation has nearly the opposite effect, strongly suggesting a critical role of N- and C-terminal charge interactions in fibril formation. These data enable us to propose structural models for the two SAA peptide amyloids, in which electrostatic interactions at the N- and C-termini underlie the structural differences between the right- and left-handed fibril types. Our findings illustrate the subtle interplay of sidechain interactions and polypeptide chain length in deciding the structural fate of an amyloid protein, and suggest competition between similar tertiary contacts (in this case, electrostatic interactions) can result in dramatic polymorphic differences between amyloids.

MATERIALS AND METHODS Materials. Peptides were custom-synthesized by Genscript (Piscataway, NJ) at >95% quoted purity; the identity and purity of all peptides were confirmed by electrospray ionization mass spectrometry (data not shown). D4-acetic acid (99 atom % D) was purchased from Aldrich (St. Louis, MO).

Deuterium oxide (D2O, 99.9 atom % D) was from Cambridge Isotope Page 5 of 37 ACS Paragon Plus Environment


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Laboratories (Andover, MA). All other chemicals were from Fisher Scientific (Pittsburgh, PA) and were of reagent grade, and were used without additional purification. Atomic Force Microscopy (AFM). Peptide samples for AFM were prepared by dissolving lyophilized peptide at 10 mg/mL in 10% v/v acetic acid and allowing a minimum of 24 hours for aggregation.31 Portions of these samples were then diluted 25- to 50-fold in water and applied to the peeled surface of a muscovite mica slab, which was then rinsed three times with water and allowed to air-dry. After drying, the surface of the mica was imaged in noncontact air-topography mode using an Asylum Research MF3-PD inverted optical AFM equipped with single-beam silicon cantilevers operating at a scan rate of 0.5 Hz and a resolution of 1050 lines per scan. Interactive flattening and plane-fitting of images was performed in the instrument software. Fibril heights were measured using the section analysis feature in the instrument software; the height of a fibril was taken as the difference in z-axis reading between the top of the fibril and the adjacent baseline, averaged over a five-pixel window. Each AFM experiment was performed on a minimum of three independent samples. Fourier Transform Infrared (FTIR) Spectroscopy.

Unless otherwise indicated,

samples for FTIR were prepared as described for AFM, but using 10% v/v D4-acetic acid in D2O. This method was chosen over that used by Rubin et al.31 to avoid complications from incomplete hydrogen-deuterium exchange. After at least 24 h fibrillization, peptide solutions were deposited on barium fluoride windows and allowed to dry in a desiccator. Spectra were then collected in transmission mode using a Perkin-Elmer Spectrum 100 spectrometer purged with dry air and equipped with a deuterated triglycine sulfate detector. All spectra are two-minute accumulations at 2 cm-1 resolution and were corrected for residual absorption of water vapor.


Second-

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derivative spectra were calculated using a 19-point window for smoothing after water vapor correction. All results shown are representative of at least three separate experiments. Raman Spectroscopy.

Samples for Raman spectroscopy were prepared exactly as

described for AFM experiments. For each sample, 5-20 µL of undiluted peptide solution was dropped onto a sheet of mica and allowed to dry in a desiccator; for most samples, this step was repeated to produce a double-thickness dried film. Spectra were acquired using a Thermo Nicolet DXR SmartRaman instrument, equipped with a 780 nm diode laser and notch filter and a high-resolution (1600 groove/mm) holographic grating (~3 cm-1 resolution).

Spectra were

collected in the backscattering (180°) geometry, using a 50 µm slit for the beam path and an incident laser power of 25-35 mW. To minimize thermal damage, samples were limited to 250 s laser exposure at a given spot; the spectra shown are averages of 3-4 such exposures. All spectra were corrected for the signal of the mica substrate, and are representative of at least three replicate experiments.

RESULTS Length-Dependent Morphology In Serum Amyloid A Fragments. Full-length SAA is a mostly α-helical protein that assembles into a homohexamer in the shape of a triangular prism, in a trimer-of-dimers arrangement.24 Within the individual SAA monomer unit, the N-terminal polypeptide segments (residues 1-12) lie entirely within an α-helix (helix 1) located at both the dimer and trimer interfaces in the hexameric assembly (Figure 1). It is thus unsurprising that this highly hydrophobic helix, nearly buried in native protein, is amyloidogenic in peptide form, as has been both predicted computationally and demonstrated experimentally.24



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Our first task was to replicate the observation that SAA1-11 and 1-12 form left-handed and right-handed amyloid fibrils, respectively, when dissolved at high concentration (10 mg/mL) in 10% v/v acetic acid.32

Peptide treated in this manner bound the amyloid-specific dye

thioflavin T (ThT), as expected (data not shown); the aggregated material as then examined by AFM (Figure 2). We observed that both peptides form amyloids up to several µm in length with greatly varying heights (thicknesses). The helicity of the fibrils matched that in the previous studies, but was less well-resolved. A few individual fibrils (upper insets in Figure 2A and 2B) showed clear left- or right-handed helical twist; in accord with the previous work, left- and right-

Figure 2: AFM images (height mode) of SAA fragment amyloid fibrils: A, SAA1-11; B, SAA1Page 8 of 37 ACS Paragon Plus Environment

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12. Scale bar in A = 3 µm (applies also to main panel of B). Insets show enlarged views of single fibrils with clear left- or right-handed periodicity, indicated by curved arrows (upper insets) or ambiguous or no periodicity (lower insets). Scale bars in insets = 100 nm. C and D: histograms of heights measured from sections through amyloid fibrils: C, SAA1-11 (n = 101, bin size = 0.25 nm); D, SAA1-12 (n = 73, bin size = 0.50 nm).

handed twist were observed only in SAA1-11 and SAA1-12, respectively.

However, the

majority of fibrils either lacked obvious periodicity or formed kinked structures in which the helical twist was ambiguous (lower insets in Figure 2A and 2B). This result probably reflects our choice of imaging method; the previous studies used high-voltage scanning electron microscopy (SEM), which produces a shaded-relief effect. Indeed, the previous authors noted that the helicity of the SAA amyloids is not easily determined when transmission electron microscopy is used in place of SEM.32 Nonetheless, the finding of expected helical character in at least a few fibrils, together with the nearly-identical infrared spectra compared to the previous work (see below), strongly suggests that the structures observed in this work match those seen previously. For this reason, we will refer to the SAA1-11 and SAA1-12 fibril morphologies as “left-handed” and “right-handed”, respectively, from this point on. An advantage of AFM is that it allows quantitative measurement of the height of features in images, allowing us to estimate the thicknesses of the amyloid fibrils. Fibril thicknesses were measured from several sections through individual fibrils of both SAA1-11 and SAA1-12, and the distributions of measured heights are shown as histograms in Figure 2C and 2D (examples of individual height traces are shown in Supporting Information Figure S1). Fibrils of SAA1-11 exhibit a near-continuum of heights, but with a peak in the distribution around 1 nm thickness



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(Figure 2C). Some fibrils were even thinner than this, with measured heights as low as 0.5 nm, comparable to the thickness of an individual β-sheet with extended sidechains; typical amyloids have diameters of 2-13 nm.1 Thus, SAA1-11 is capable of forming unusually narrow amyloid fibrils. Thicker fibrils, however, were also observed, suggesting the thinner SAA1-11 fibrils assemble into bundles, as seen in some AFM images (Figure S1).

SAA1-12, in contrast,

invariably formed thicker fibrils (Figure 2D), mostly 2-3 nm in cross-section (with a peak at 2.5 nm); fibrils thinner than 2 nm were rare, and fibrils less than 1.5 nm across were not observed. Like the shorter peptide, SAA1-12 fibrils also associated into bundles with thicknesses up to 10 nm in some cases. In summary, the AFM data reiterate the observation that SAA1-11 forms lefthanded amyloids while SAA1-12 forms right-handed amyloids, but furthermore show that fibrils of the latter are generally thicker than those of the former—specifically, the lower limit of fibril cross-section is much greater in SAA1-12 than in SAA1-11. Vibrational Spectroscopy Reveals Secondary Structural Differences between SAA Fragments. We next turned our attention to the secondary and tertiary structural interactions within the fibrils, as revealed by FTIR and Raman spectroscopy. FTIR spectra of dried films of amyloid, in the structure-sensitive amide I’ region, are in good agreement with those reported previously31 (Figure S2). SAA1-11 and SAA1-12 fibrils exhibit peaks at 1690 and ~1620 cm-1, both of which are indicative of β-sheet secondary structure.33,34 In our samples, another band was observed at 1735 cm-1, which we assign to residual acetic acid in the sample. The lowerfrequency β-band was broad in SAA1-11 and sharper for SAA1-12, as in the previous study.31 We therefore calculated second derivatives of the spectra in Figure S2A to reveal the presence of any overlapping component bands. The results (Figure S2B) show that SAA1-12 is dominated by a single β-sheet peak at 1618 cm-1 (with minor side-bands at 1605 and 1632 cm-1), while


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SAA1-11 exhibits at least two sub-peaks with frequencies of 1628 and 1622 cm-1. In addition, both fragments also displayed a sharp peak with a minimum at 1689 cm-1 (the high-frequency βsheet component), the acetic acid peak at 1738 cm-1, a variable peak at ~1713 cm-1 probably arising from protonated Glu or Asp sidechains,35 and a weak band at 1660 cm-1, which likely represents disordered structure36–38 and is more intense in SAA1-12. To gain further information about the nature of the structural differences between the two amyloids, we performed Raman spectroscopy on dried films of the SAA fragment amyloids (Figure 3). Raman spectroscopy provides access to a broader range of protein vibrational modes than FTIR39 and thus is potentially more structurally informative, despite its inherently lower



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Figure 3: Dispersive Raman spectra (780 nm excitation) of dried films of SAA1-11 (black) and SAA1-12 (gray).

A, full spectrum from 500-1760 cm-1 with general assignments given

(complete assignments in Table S1). Peaks marked with an asterisk (*) are residual peaks from the mica used as substrate. B, expanded view of the structure-sensitive 1100-1700 cm-1 spectral region, with peaks differentiating the two peptides indicated. See text for explanation.

sensitivity. Raman spectra have proven invaluable in elucidating structural features of other amyloid proteins.40–44 Dispersive Raman spectra (780 nm excitation) of SAA amyloid films are shown in Figure 3A; assignments of peaks in the 500-1700 cm-1 spectral region are given in Table S1. Most substantial differences between the two peptides occur in the 1100-1450 cm-1 spectral range; this region, along with the structure-sensitive amide I region, is shown in expanded view in Figure 3B. Both peptides display a remarkably narrow amide I peak with a maximum at 1671 ± 1 cm-1, indicative of β-sheet secondary structure. Four peaks differentiate the Raman spectra of SAA1-11 and SAA1-12; three of these peaks lie in the conformation-sensitive amide III region between ~1200 and 1350 cm-1 (Figure 3B). Peaks in the lower-frequency end of this range are indicative of β-structure.39,45–47 In SAA1-11, the lowest-frequency amide III maximum appears at 1240 cm-1, while that of SAA112 lies at 1233 cm-1. This significant shift reflects differences in the geometry of the β-sheets, specifically the backbone dihedral angles46,47 (discussed below).

SAA1-12 also exhibits a

pronounced shoulder at 1254 cm-1 that is much weaker in SAA1-11; this band most likely reflects disordered structure.39,48 More significantly, SAA1-12 displays a pronounced peak at 1283 cm-1 that is very weak in SAA1-11.

This unexpected feature is consistent with α-helix


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secondary structure,39 and suggests that SAA1-12 contains come helical character, even though no α-helix component is evident in the amide I/I’ peak in either Raman or FTIR spectra. This apparent discrepancy between amide I and III peaks can be explained by the different physical mechanisms underlying the structural sensitivity of these two vibrational modes. The amide I mode is primarily a C=O stretching vibration,33,34,49 and thus is sensitive to hydrogen bonding, which produces correlation between C=O oscillators throughout a given secondary structure element.49 The amide III mode, in contrast, is mainly a C-N stretching vibration that is sensitive to local geometry, particularly the ψ dihedral angle, at each individual residue.46,50,51 Thus, the amide III mode is a more local probe of polypeptide structure, while the amide I band is more global. The appearance of an α-helical signal in the amide III but not amide I/I’ band, therefore, suggests the presence of a significant number of residues with αhelical geometry, but without the intrachain hydrogen bonding of an extended α-helix. The fourth difference between the SAA1-11 and SAA1-12 Raman spectra is a pronounced broad band centered at 1404 cm-1 in SAA1-12, which is essentially absent in SAA111 (Figure 3B). This peak is difficult to assign with any certainty. While it could reflect the structure-sensitive amide S (Cα-H bending) mode52 or the symmetric carboxylate stretch53 of the Asp12 sidechain, we consider these assignments unlikely because amide S is structure-sensitive only with resonance enhancement54 (and would thus be similar for similarly-sized peptides in the absence of resonance), and because few deprotonated carboxylates would occur at the pH (~3) of this experiment (furthermore, carboxylate stretching modes are weak in the Raman). Instead, we believe the 1404 cm-1 band arises from alkyl groups on amino acid sidechains, most likely Leu, Ala, Ser, or Gly, all of which exhibit peaks in this region.55–57 Interestingly, we note that a similar, unassigned peak at this position was seen in Raman spectra of β-lactoglobulin, and



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disappeared upon thermal denaturation of that β-sheet protein,58 suggesting some environmental sensitivity.

We therefore believe this peak reflects a difference in the conformation or

environment of one or more sidechains in SAA1-12 relative to the corresponding residues in SAA1-11. Role of Electrically-charged Groups in SAA Fragment Fibril Formation and Polymorphism.

Studies of full-length SAA1.1 have shown that electrostatic interactions

involving the first twelve residues mediate the oligomer stability of the SAA1.1 hexamer, and (in the case of Arg1) the ability of SAA1.1 to bind glycosaminoglycans24 and lipoproteins.59 This finding raises the possibility that charge interactions underlie amyloidogenesis by N-terminal SAA peptides, and may play a role in the distinct morphologies formed by SAA1-11 and SAA112. At the pH of our experiments (~3), both SAA fragments will have three predominantly charged groups: the N- and C-termini and the sidechain of Arg1 (the Glu9 and Asp12 sidechains would be only about 10% deprotonated at this pH). To individually examine the roles of these charged groups in SAA fibrillization, we constructed peptide variants in which either the N- or C-terminus (but not both) was acetylated or amidated, respectively, to eliminate the electrical charge. The acetylated and amidated peptides were then incubated in 10% v/v acetic acid and subject to the experiments described above.

Table 1. Summary of Fibril Characteristics for SAA1-11 and SAA1-12 Variants Peptide Morphology (AFM) Amide I’ (FTIR)a, cm-1 Amide III (Raman), cm-1 SAA1-11 Left-handed fibrils 1621, 1628, 1690 1240 NAcSAA1-11 Left-handed fibrils 1620, 1628, 1690 1242 SAA1-11-NH2 Globules, few fibrils 1619, 1665, 1689 1238 SAA1-11 R1Q Amorphous aggregate 1621, 1668, 1689 1225, 1252 SAA1-11 E9Q Right-handed fibrils 1622, 1633, 1663, 1694 1257 SAA1-12 Right-handed fibrils 1621, 1661, 1690 1233, 1254, 1283 NAcSAA1-12 Amorphous aggregate, 1623, 1672 1239 very few fibrils SAA1-12-NH2 Fibrilsb 1621, 1665, 1689 1237, 1254, 1284 Page 14 of 37 ACS Paragon Plus Environment

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An overview of amyloid structural features for SAA1-11 and SAA1-12 variants. aAmide I’ maxima taken from minima in second-derivative spectra. bSAA1-12-NH2 had dimensions consistent with those of SAA1-12 but lacked obvious periodicity, so handedness is not indicated. The results of these experiments are shown in Figure 4 and summarized in Table 1. All N- and C-terminally blocked peptides formed aggregates with some degree of ThT binding (data not shown), but not all of these peptides formed amyloid fibrils: briefly, N-acetylation had little effect on SAA1-11 while abolishing fibrillization of SAA1-12, whereas C-amidation had nearly the opposite effect. N-acetyl SAA1-11 (NAcSAA1-11) forms fibrils with a left-handed helical twist, like wild-type SAA1-11 (Figure 4A). Likewise, the FTIR spectra of NAcSAA1-11 show the same distinctive broad β-sheet band, with components at 1622 and 1628 cm-1, as seen in wild-type SAA1-11. Raman spectra, furthermore, show the amide III maximum near 1240 cm-1, similar to that seen in the unblocked peptide. NAcSAA1-11 did display a weak band at 1296 cm1

that was not evident in the unblocked peptide (Figure 4A, bottom panel), but this peak was also

seen in NAcSAA1-12 and likely reflects the amide III signal of the N-terminal acetylation. We thus conclude that N-terminal blockage does not appreciably affect fibril formation or structure in SAA1-11. On the other hand, N-terminal acetylation effectively blocked fibril formation by SAA112. AFM images of this peptide, collected 48 h after preparation, reveal mostly amorphous clumps with a few thin, short fibrils in some fields (Figure 4B); this figure shows one of the very few such fibrils seen. While all other peptide solutions converted to a viscous semi-solid gel within 24 hours of preparation (due to formation of an interpenetrating network of fibrils), NAcSAA1-12 remained fluid, with little change in viscosity, for over a month after preparation



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Figure 4: Structural analysis of N-acetylated and C-amidated variants of SAA1-11 and SAA112: (A) NAcSAA1-11; (B) NAcSAA1-12; (C) SAA1-11-NH2; (D) SAA1-12-NH2. Each panel shows (clockwise, from upper left) height-mode AFM images of fibrils, FTIR spectra (amide I’ band), second-derivative of the amide I’ region, and Raman spectrum. Scale bars = 500 nm in C, 300 nm in other panels. *Residual band from mica substrate.


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(data not shown). The FTIR spectrum (Figure 4B, top right) was dominated by the broad peak around 1550 cm-1 (an overlap of antisymmetric carboxylate stretch, residual amide II, and HDO bending modes, extending off-scale in the figure) that is only a shoulder in the other samples’ spectra. The weak amide I’ peak appeared at 1670 cm-1, suggestive of turns,33 with a secondary β-sheet peak at 1624 cm-1.

The Raman spectrum of NAcSAA1-12 lacked the amide III

components at 1252 and 1283 cm-1 and the shoulder at 1404 cm-1 (all seen for unblocked SAA112).

It therefore appears that N-terminal acetylation virtually prohibits fibril formation by

SAA1-12, and instead results in amorphous aggregate with some turn and residual β-structure. C-terminal amidation, unlike N-terminal acetylation, had a larger effect on SAA1-11 than on SAA1-12. Solutions of SAA1-11-NH2 exhibited delayed fibrillogenesis compared to the other peptides; the solutions became gel-like only after several days of incubation (data not shown). AFM images collected after 74 h show the presence of fibrils of indistinct periodicity (Figure 4C), along with many globular amorphous particles (not shown).

The fibrils had

thicknesses of less than 1 nm in many cases (data not shown), suggesting a similarity to those of wild-type SAA1-11. FTIR spectra showed a broad peak centered at 1665 cm-1 (unstructured) and a peak at 1621 cm-1 (β-sheet) whose intensity was lower than that of SAA1-11, consistent with a lower overall fibril content. Second-derivative analysis reveals the major β-peak to consist of a single component, while the Raman spectrum of SAA1-11-NH2 was similar to, but weaker than, that of unblocked SAA1-11.

We conclude that C-terminal amidation greatly

disfavors but does not abolish fibril formation by SAA1-11, and has minor effects on secondary structure. C-terminally amidated SAA1-12, in contrast, was nearly indistinguishable from ordinary SAA1-12 (Figure 4D). Fibrils with a minimum thickness of 1.5 nm were observed in AFM



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images (though no periodicity was evident), and the FTIR and Raman spectra are nearly identical to those of unblocked SAA1-12 (compare to Figure 3 and S2). Notably, the Raman spectral features at ~1250 and 1283 cm-1 characteristic of the wild-type peptide also appear in SAA1-12NH2, as does the unidentified shoulder above 1400 cm-1 (though in SAA1-12-NH2 this feature is shifted up somewhat, to 1410 cm-1). In summary, we conclude that N-terminal blockage has little effect on fibril formation or morphology in SAA1-11, but essentially abolishes fibrillization of SAA1-12.

C-terminal

amidation, in contrast, inhibits SAA1-11 fibrillization (with small effects on fibril conformation) but has no appreciable effect on SAA1-12 amyloidogenesis.

Taken together, these results

strongly suggest that the left-handed structure of SAA1-11 depends on an interaction involving the free (charged) C-terminus, while the right-handed SAA1-12 fibril structure requires an interaction at the charged N-terminus. In a parallel set of experiments, we also explored the role of electrostatic interactions involving the two charged sidechains of the SAA fragments, Arg1 and Glu9 (the Asp12 residue of SAA1-12 has already been shown nonessential to formation of the right-handed fibril structure31). To this end, SAA1-11 variants were constructed in which these residues were replaced with a similarly-sized, uncharged Gln residue, resulting in R1Q and E9Q mutants of SAA1-11. Amyloids of these peptides were then examined as described above. The results (Figure S3 and Table 1), however, were less conclusive than seen with the terminally-blocked peptides. Briefly, R1Q SAA1-11 formed only globular amorphous aggregates but displayed FTIR and Raman spectra similar to wild-type SAA1-11, except for a broader amide I band in Raman spectra, suggestive of a distribution of conformations. E9Q SAA1-11 formed fibrils with a uniform ~2 nm cross-section (data not shown) and a tendency to self-coil in a right-handed


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fashion (Figure S3B, top left). The FTIR spectrum contained a two-component β-sheet signal, and the Raman spectrum showed a very unusual amide III band profile localized mostly between 1250-1260 cm-1. This latter signal is suggestive of disordered structure, polyproline-II structure, or type III β-turns50,51, though we note that the amide III peak was quite variable from sample to sample for this peptide (typical results are shown), suggesting considerable structural heterogeneity (data not shown). It is most likely that the addition of Gln opened new pathways of fibrillization to the peptide, as this amino acid is amyloidogenic due to its strong propensity to form polar zippers.3,60,61 Because of the ambiguity of these results, Gln mutagenesis was not attempted on SAA1-12.

DISCUSSION New Insight into the Structure of SAA1-11 and SAA1-12 Amyloids. The observation that extension of SAA1-11 by a single amino acid can change the resulting amyloid fibrils from left- to right-handed31,32 provided one of the most striking examples of amyloid polymorphism reported to date. This finding not only has intriguing implications for the mechanism of amyloid formation (since most natural amyloids are left-handed), but illustrates the frustrating complexity of predicting or modeling amyloid structures based on sequence similarity to amyloids of known structure, or of inferring pathological or materials properties associated with amyloids of a given polypeptide sequence.62 The present results bring some clarity to the issue of SAA fragment polymorphism by highlighting some key structural differences between the two types of fibrils. The thickness of the thinnest SAA1-11 fibrils approximates that of a single β-sheet (measured perpendicular to the plane of the sheet), suggesting the smallest SAA1-11 fibrils consist of a single protofilament. Page 19 of 37 ACS Paragon Plus Environment


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This is a remarkable observation, as isolated β-sheets lacking tertiary contacts with other secondary structure elements are quite rare. When such sheets do occur, they are stabilized by cross-strand interactions between sidechains, as in monomeric β-hairpin peptides.63–67 However, the observation of thicker SAA1-11 fibrils (Figure 2) indicates that higher-order assembly of SAA1-11 protofilaments can occur, presumably by stacking of sheets.3,60 Furthermore, we have found that N-terminal acetylation of SAA1-11 does not affect fibrillogenesis or structure, while C-terminal amidation inhibits fibril formation and results in fibrils containing a greater proportion of disordered secondary structure compared to β-structure. This result suggests that a free C-terminus is essential to the left-handed SAA1-11 fibril structure. However, the presence of Arg1 also appears essential for this structure: in previous work, N-terminal truncation of SAA1-12, forming SAA2-12 (which has the same length as SAA1-11), results in right-handed fibrils.31 Thus, both Arg1 and a free C-terminus are needed for the left-handed fibril structure in the SAA peptides. Our observations enable us to propose a structural model for SAA1-11 fibrils that accounts for both the stability of the single-sheet fibrils and the necessity of Arg1 and the Cterminus in fibril formation.

We suggest that SAA1-11 protofilaments consist of a single

antiparallel β-sheet in which individual molecules form single strands with an N-terminal overhang consisting at least of Arg1 (Figure 5A). Though antiparallel sheets are relatively uncommon in amyloids, this architecture would juxtapose the C-terminus and Arg1 residues of


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Figure 5: Proposed models for SAA1-11 and SAA1-12 amyloid fibrils based on the present data. A, antiparallel protofilament of SAA1-11, stabilized by electrostatic interactions between Arg1 and the C-terminus of the neighboring strand (red arrows). Orientations of other sidechains are arbitrary. B, right-handed parallel β-helix model for SAA1-12, in which successive turns are stabilized by interactions between N- and C-termini. This model is based on the crystal structure of the Tenebrio molitor antifreeze protein (PDB code 1ezg). Residues flanking the β-strand have right- and left-handed α-helical ϕ/ψ angles, labeled αR and αL respectively. The relative positions of these features within the peptide sequence cannot be determined from the present data and should be considered arbitrary. See text for details. Figure generated using PyMOL 68. neighboring strands, allowing for cross-strand ionic interactions at the edges of the sheet (red arrows in Figure 5A). The stability of the isolated protofilament is presumably due to the presence of two such interactions per strand and to their location at the strand ends, preventing unzipping of the sheet. The lowered extent of fibril formation by SAA1-11-NH2 (Figure 4C and



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Table 1) may be due to a lack of this charge interaction, destabilizing the protofilament. Fibrils of this architecture would display two Phe sidechains on either side of each strand, allowing for formation of complementary aromatic (CH-π or π-stacking) interactions between protofilaments, allowing multiple filaments to stack together, explaining the occurrence of thicker fibrils. The latter effect may also explain the variable-proportion double β-sheet maximum in the FTIR spectra, since the position of β-sheet maxima in the amide I’ band depends in part on sheet size and on solvent exposure.33 In this model the N-terminus does not form any appreciable contacts, accounting for the lack of an effect of N-terminal acetylation on SAA1-11 fibril formation or structure. A structural model for SAA1-12 fibrils, in contrast, needs to account for: 1) the righthanded morphology, 2) an apparent insensitivity to the identity of residue 12 or N-terminal truncation,31 3) the presence of both disordered/loop and helical secondary structure, as seen in the amide III Raman band (Figure 3), and 4) a requirement for a free N-terminus (Figure 4). We will address each of these experimental constraints separately. There are at least three peptide structural motifs that could produce the right-handed twist of the fibrils. The first of these is a rare left-handed α-helical crossover between adjacent parallel β-strands.69 This structure can be immediately ruled out because SAA1-12 is too short to form two stable β-strands with an intervening α-helix. A second possibility is a strand-turn-strand motif with a positive “stagger” (N-terminal longer than C-terminal strand, resulting in an overhang).70 This structure would produce fibrils with dimensions consistent with AFM images; it also explains why the shorter SAA1-11 cannot form right-handed fibrils (the C-terminal strand must be long enough to be stable), and why deletion of Arg1 is compatible with right-handed structure (a positive stagger would remain, so long as the N-terminal overhang was 2 or more


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residues in length). However, this model cannot explain why N-terminal acetylation abolishes SAA1-12 fibrillization (since the N-terminus would not form any contacts), nor does it explain the presence of α-helical signal in the Raman spectra. The third possible structural model for SAA1-12 fibrils is a right-handed parallel β-helix. Though unusual, natural β-helical amyloids have been observed, such as in the fungal prion protein HET-s(218-289).71 Right-handed β-helices, such as the well-known p22 phage tailspike protein72 and the pentapeptide repeat proteins73 usually contain 16-34 residues per turn of the helix,74 but examples with fewer residues per turn are known. For example, the antifreeze protein from the beetle Tenebrio molitor exhibits 12 residues per turn of the β-helix, with a helical cross-section of ≈ 1.4 nm (not including sidechains).75 Since the 12-residue SAA1-12 fibrils have a similar cross-section in AFM images, we considered the possibility that SAA1-12 forms right-handed β-helices like the T. molitor antifreeze protein. The antifreeze protein is stabilized by sidechain interactions and disulfide bridges in the helix interior.75 SAA1-12 contains a large number of hydrophobic residues, including 4 Phe (Figure 1). In a thorough statistical survey of both left- and right-handed β-helical protein structures, Iengar et al.74 showed that phenylalanine (along with other large hydrophobic amino acids) shows a marked preference for the interior of right-handed (but not left-handed) β-helices, allowing these structures to be stabilized by hydrophobic and/or aromatic interactions. Such internal hydrophobic contacts are responsible for the stability of the aforementioned HET-s(218289) amyloid.71 More importantly, these same authors showed that successive turns of right-handed βhelices generally observe the following sequence of structural repeats: Strand 1 – (αL) – Strand 2 – (αL – Loop – αR) – Strand 3 – (αL/αR – Loop – αL) …



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in which αL and αR represent single residues with left- or right-handed α-helical ϕ/ψ angle combinations, respectively, and “Loop” represents loop segments of varying length and sequence (some short helical turns lack one or two strands, as in the antifreeze protein, whose structural repeat pattern is Loop – αR – Strand – αL).75 Thus, right-handed β-helices invariably contain both residues with right-handed α-helical conformation and loops. This is exactly what is seen in the Raman spectrum of SAA1-12, which contains a shoulder at 1254 cm-1 (consistent with loops or disordered structure) and a peak at 1282 cm-1 (α-helix), along with a typical β-sheet band at 1234 cm-1 (Figure 3 and Table 1). According to the semi-empirical formula developed by Mikhonin et al.46,51 for correlation of the amide III frequency νAmIII with the ψ Ramachandran angle, the left-handed α-helical residues would give rise to νAmIII in the 1190-1210 cm-1 region and would thus be obscured by the very strong Phe peak in this area. As explained above, the absence of α-helical signal in the FTIR amide I’ band is due to the presence of individual residues with helix-like ϕ/ψ angles, rather than extended helices. While the presence of a highfrequency (1690 cm-1) β-component in the amide I’ FTIR band (Figure S2) might be considered inconsistent with parallel β-sheets, this feature is not, in fact, unique to antiparallel sheets and can arise in highly extended sheets of either parallel or antiparallel arrangement.76 Notably, the HET-s(218-289) amyloid, known to be a parallel β-helix, exhibits a high-frequency component in its FTIR spectrum.13 We therefore believe that SAA1-12 forms right-handed β-helices stabilized in part by Phe sidechain interactions, likely in the helix interior. This model alone cannot, however, explain the need for an unblocked N-terminus for formation of SAA1-12 fibrils. However, if each peptide chain forms one complete turn of the helix, the N- and C-termini of successive peptides would align, stabilized by a salt bridge interaction (Figure 5B) as has previously been engineered into


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designed β-helical amyloid materials.77 This charge interaction would be impossible with an acetylated N-terminus, while with an amidated C-terminus, a nearly equivalent salt bridge interaction could be formed with the small Asp12 sidechain, requiring only a minor local bond rotation. In N-acetylated peptide, such a “rescuing” sidechain interaction (between Arg1 and the free C-terminus) would be entropically unlikely, due to the tight curvature of the β-helix and the large size of the Arg sidechain; thus, N-acetyl SAA1-12 is effectively non-amyloidogenic. These structural models suggest that lengthening the SAA fragment from 11 to 12 residues is sufficient to tip the balance of electrostatic interactions from favoring an antiparallel Arg1-C-terminus interaction (as in SAA1-11 fibrils) to formation of a right-handed β-helix stabilized by a combination of electrostatic interactions between N and C termini and by sidechain packing in the helix interior. The increased favorability of the latter in SAA1-12 cannot be entirely due to length, however, as SAA2-12 (also 11 residues) forms right-handed amyloids.31 Thus, Arg1 serves as a “gatekeeper” in selecting which fibril structure will form: when it is present, an intermolecular salt bridge with the C-terminus of another peptide dominates in shorter peptides, whereas the added stability of a twelfth residue in each β-helical turn overwhelms this effect in longer peptides. In the absence of Arg1, the helical structure is the only possibility (assuming the right-handed fibrils formed by SAA2-12 are β-helical like SAA1-12). Why does N-acetyl SAA1-12 not form thin left-handed fibrils like SAA1-11?

We

speculate that in the absence of a charged N-terminus, the C-terminus (or possibly Asp12) forms an intramolecular salt bridge with the Arg1 sidechain. Such a hairpin-like conformation has been observed in amyloids of β2-microglobulin,78 but this arrangement would prohibit formation of the extended antiparallel strands as suggested for the SAA1-11 fibrils (and would be



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unfavorable for SAA1-11 due to insufficient length). The FTIR amide I’ maximum at 1670 cm-1 for NAc-SAA1-12 (Figure 4B) is, in fact, suggestive of turns, and the β-sheet signals in amide I’ and III bands support our hypothesis of hairpin formation. Moreover, sequence analysis of SAA1-12 shows a marked propensity for turn formation centered on the –FLGE- segment, according to the NetTurnP algorithm79 (data not shown). The literature contains numerous examples of monomeric β-hairpins stabilized by cross-strand sidechain interactions such as those proposed for NAc-SAA1-12.63,65,66 However, further work will be needed to conclusively test this model.

CONCLUSIONS We have shown that the previously mysterious ability of SAA N-terminal peptides to form amyloids with right-handed/left-handed helical polymorphism can be readily explained by a structural model in which competing electrostatic interactions at the C-terminus, involving either the Arg1 sidechain or the N-terminus, decide between left-handed or right-handed morphology (respectively). This model is fully consistent with the spectroscopic data provided here and accounts for the observed sensitivity of fibrillization to blockage of the N- or C-termini. Since the N-terminus is critical to full-length SAA amyloidogenesis, these results suggest differing charge interactions may be involved in distinct pathologies of AA amyloidosis. Furthermore, since N- and C-terminal charge interactions have been exploited in the design of stable peptide nanotubes and bilayers,77,80 our results may inform further work on protein nanomaterial design in which charge interactions can be manipulated in order to alter material properties.

Acknowledgements Page 26 of 37 ACS Paragon Plus Environment

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We are grateful to Dr. Emily Fogle for access to and assistance with PyMOL, to Dr. Derek Gragson for assistance with AFM and light scattering measurements, and to Jeffrey Shen for conducting light scattering experiments. This work was supported by Cal Poly College of Science and Mathematics startup funds (to E. M. J.) and a College of Science and Mathematics Frost Undergraduate Research Fellowship (to J. M. J.).

Supporting Information: Example AFM height traces, FTIR spectra of SAA amyloids, table of assignments of Raman peaks, AFM and spectroscopic data for R1Q and E9Q SAA1-11 peptides. This material is available free of charge via the Internet at http://pubs.acs.org.

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