This journal is©The Royal Society of Chemistry 2018 Chem. Commun.

Cite this:DOI: 10.1039/c8cc06528d

Self-assembly of penta-selenopeptides intoamyloid fibrils†

Ram P. Gokula, Jaladhar Mahato, Harkesh B. Singh * andArindam Chowdhury *

Here, we report the synthesis of a penta-selenopeptide consisting

of five benzyl protected selenocysteine residues. This selenopeptide

was well characterized by both one- and two-dimensional (D) NMR

spectroscopies. We find that the solution conformation is enriched

with b-sheet structures, which have a propensity to self-assemble

and form amyloid fibrils.

The main source of selenium (Se) in biological systems is thenaturally occurring amino acid selenocysteine (Sec), which isfrequently found in the active sites of selenoproteins likeglutathione peroxidases, iodothyronine deiodinases, thioredoxinreductases, formate dehydrogenase H, etc., where it plays severalcomplex roles.1–6 On the other hand, synthetic selenopeptidesare an interesting class of compounds that have been utilized forseveral applications, such as in probing the structure–activityrelationship of proteins,7,8 designing drug scaffolds9 and testingthe specificity of enzymes.10

The structural analysis of peptides is expected to illustratepeptide–peptide and protein–peptide interactions, which provideinsight into the design of new classes of peptides with certainbiological relevance.11 However, there is a dearth of the literatureon the structural characterization and purity of selenopeptides,especially using NMR spectroscopy.12–14 It should be mentionedthat structural analyses are restricted for Sec–Sec coupledpeptides due to the lack of properly resolved NMR spectra,which in turn limits further exploration of selenopeptides forvarious morphological and biological investigations.

It is well known that peptides of diverse lengths and sequencesundergo self-assembly into insoluble, mesoscale aggregates suchas amyloids,15–17 the accumulation and deposition of whichare associated with several neurodegenerative diseases suchas Alzheimer’s and Parkinson’s.18,19 Although a wide range ofpolypeptides form amyloid fibrils, they exhibit very similarbiophysical properties owing to the presence of a common

cross b-spine structural motif, a set of b-strands orientedperpendicularly to the fibrillar axis.11,20,21 Several approachesexist that favour peptide self-assembly, such as the use ofaromatic N-capping agents,22 complementary charges,23 changein amino acid chirality,24 and halogenation.25,26 Phenylalanine(Phe) plays a special role in driving peptide self-assembly asdemonstrated by the wide use of the Phe–Phe motif in variousordered assemblies.27 While longer polypeptides readily involvein amyloid fibril formation, smaller peptides enriched witharomatic residues are also capable of forming functionalamyloids,28,29 some of which can be used for drug delivery,tissue engineering, and biosensing.20

Recently, several studies have demonstrated that seleniumin its different forms, alone or combined with Vitamin E, mayhelp in preventing Alzheimer’s pathology.30 In particular, it hasbeen shown that seleno-L-methionine can reduce amyloid-bdeposition in cell culture and in a transgenic mouse model.31

In this context, it is important to know whether selenopeptidescan also form amyloids, similar to their natural counterparts.However, as of yet, there has been no report of amyloid fibrilformation based on the aggregation of selenopeptides.

It is reported that the coupling efficiency of Sec to formchains of amino acids is as high as that of the cysteine (Cys)analogue;32 however, coupling of Sec to form a chain of five Secunits has not yet been reported in the literature. It is alsowell known that selenium is involved in intra-/intermolecularSe� � �Se, Se� � �N, Se� � �O, and Se� � �H interactions. This motivatedus to synthesize a new sequence of selenopeptides that can playa remarkable role in the determination of their morphologicalproperties.33–35 In this work, we report the synthesis of a penta-selenopeptide, prepared by solid phase peptide synthesis(SPPS) with a rink amide resin as a solid support. Further, weestablish that the penta-selenopeptide forms amyloid fibrils viaself-assembly, very similar to naturally occurring peptides suchas pentaphenylalanine.36 The structure and morphology of thispeptide and its self-assembly have been characterized exten-sively using a variety of biophysical methods, both in solutionand in the solid state.

Department of Chemistry, Indian Institute of Technology Bombay, Powai,

Mumbai 400076, India. E-mail: chhbsia@iitb.ac.in, arindam@chem.iitb.ac.in

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc06528d

Received 10th August 2018,Accepted 13th September 2018

DOI: 10.1039/c8cc06528d

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Peptide 1 was synthesized by SPPS using Fmoc-Sec-(Bzl)-OHas a precursor [Scheme 1].32 The details of the synthesis andsample preparation procedure are provided in the ESI.† It iswell known that Sec in the selenol (SeH) form is highly unstableand hence has to be protected. Benzyl protected Sec has beenused for the synthesis of selenopeptides, which requires harshconditions to deprotect to generate SeH and is considered as alimitation for selenopeptide chemistry.32 Here we use thislimitation as an advantage to synthesize peptide 1. The primarystructure of the synthesized peptide was extensively characterizedby high resolution mass spectrometry (HRMS), and 1D [1H,13C and 77Se] and 2D [COSY, NOESY, and TOCSY] NMR spectro-scopy. The 1H, 77Se, 13C, 1H–1H COSY and TOCSY NMR spectraand assignments of peptide 1 are given in the ESI.† Here itshould be noted that the NOESY spectrum shows one spatialinteraction between type a and h protons, which suggests theformation of secondary structures in the solution (Fig. 1).

To test whether peptide 1 forms a secondary structure insolution, we performed FT-IR and CD spectroscopy in aceto-nitrile solution. The FT-IR spectrum (Fig. 2a) shows the presenceof b-sheet content as evident from the amide I maximum atB1634 cm�1.20 The CD spectrum (Fig. 2b) changes with varia-tion of concentration (Fig. S11a, ESI†) and indicates the presenceof b-sheets along with other secondary structures. It should bepointed out that the solution of peptide 1 in acetonitrile wasquite turbid, indicating the presence of colloidal suspensions.The high b-sheet propensity along with the lack of a transparent

solution suggested that amyloid like fibrils may form in thesolution.37 To confirm whether that is so, we subjected peptide 1to further tests for amyloid formation in solution.

It is well known that a large number of molecules showspecific changes in absorption and emission spectra in terms ofboth transition energy and enhanced fluorescence, upon bindingwith amyloid fibrils.38 Historically, amyloid formation wasdetected in terms of an enhanced fluorescence of benzothiazoledye Thioflavin-T (ThT) upon binding with the fibrils, which isexpected to significantly enhance the emission intensity uponbinding to the cross-b structure of amyloid fibrils. We find thatthe fluorescence emission (Fig. 2c) was enhanced upon additionof selenopeptide colloidal solution (Fig. S11b, ESI†), whichindicates the presence of a cross b-spine structural motif, asignature of amyloid formation. It should be pointed out thatthere was no significant enhancement of ThT fluorescence below20 mM peptide. This is consistent with the high population ofother secondary structures at lower concentrations of peptide 1as observed in the CD spectrum. However, it is difficult tocomment on whether the enhancement of ThT fluorescenceoccurs due to the formation of amyloid oligomers (assembly offew or few tens of 1) or whether the formation of extendedfibrils, as both have the common cross-b structural motif.38–40

To understand the morphology, optical and electron micro-scopies were performed by casting the fibrils from solution on asubstrate. Morphological analyses were performed in the driedstate and the drying process might have affected the observa-tions. Fig. 3a shows an epi-fluorescence microscopy image of300 mM peptide 1 and 250 mM ThT on glass coverslips dropcastfrom acetonitrile solution. This reveals the presence of spatiallyseparated fibrillar aggregates with lengths varying from a few totens of micrometers. Importantly, the high contrast in theseimages clearly indicates that the augmented fluorescence (Fig. 2c)is due to the formation of extended amyloid fibrils as opposed tooligomers. Further, we find that the widths of the fibrils obtainedby fluorescence microscopy are often beyond the diffractionlimit (200–500 nm). These findings indicate the formation ofhigher order aggregates consisting of several entwined amyloidfilaments, similar to that observed for natural amyloid fibrils.19

Using optical microscopy, it is challenging to estimate the actualdiameters of individual amyloid fibrils, which are expected torange from 7 to 15 nm.21,29 Therefore, we performed HR-TEMand AFM of peptide 1 in acetonitrile (0.25 mM) (Fig. 3b and c).The TEM image (Fig. 3b and Fig S12, ESI†) shows the presence offew discrete fibrils with B12–14 nm diameter, while the vastmajority of fibrils have much larger widths (B30–300 nm) due tothe formation of laterally associated aggregates. The formationof higher order aggregates is further supported by the AFMimage (Fig. 3c), where the fibrillar widths are typically morethan 20 nm. Thus, Fig. 3a–c reveal that peptide 1 formsunbranched higher order extremely long fibrils similar to thosereported for functional amyloids.13,28 To understand the detailsof internal molecular arrangement and the nature of the cross-bstructure of the filaments that constitute the higher order fibrils,we performed X-ray fiber diffraction measurements of peptide 1.This shows the presence of three prominent reflections (Fig. 3d).

Scheme 1 Synthesis of a penta-selenopeptide using Fmoc-Sec (Bzl)-OHas a precursor.

Fig. 1 NOESY NMR spectra of peptide 1.

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Here, the reflection at B4.2 Å is due to the hydrogen bondingdistance between b-strands, while the 7 Å reflection correspondsto the repetitive polypeptide chain direction and the 15 Å reflec-tion arises from the repetition of b-sheets with the strandsrunning perpendicularly to the fibril axis. The characteristic

X-ray pattern indicates the presence of a common cross b-spinestructure in the self-assembly of peptide 1.21 Although the 15 Åreflection is higher than the normal 11 Å distance found innaturally occurring amyloid b fibrils, this is not surprising as thepresence of extra one carbon and one Se atom in the penta-selenopeptide can increase the 4 Å distance between the twob-sheets and this peak is strong due to the lateral aggregation of avast majority of protofilaments (as evidenced in TEM and AFM).It is well known that amyloid fibrils are made up of entwinedfilaments. To understand the extent of entwining of the peptide 1filaments, we performed FEG-SEM measurements at 40 mM.The twist of individual fibrils can be seen in the SEM image(Fig. 3e) and we found that the half helical pitch length isB250 nm with B25 nm width. The presence of both paralleland entwined fibrils with different widths in both SEM and TEMimages is reminiscent of the polymorphism of the amyloidstructure.20,21

To reveal the molecular details of ThT binding to peptide 1amyloid aggregates and to understand the arrangement orcoiling of the protofilaments which constitute the fibrils, weperformed fluorescence anisotropy imaging by circularly polarizedexcitation and collected the emission in orthogonally polarizedchannels (ESI†).41 It is well known that ThT binds to the proto-filament along the protofilament axis.38 Fig. 3f shows the maxi-mum anisotropy image of ThT bonded amyloid fibril(s) obtainedfrom the maximum projection of images collected by polarization-resolved fluorescence microscopy at 101 consecutive analyzerangle intervals (Fig. S14a, ESI†). It is clear from the image thatthe anisotropy value (r) is almost the same for all three fibrils. Thelow r value suggests that although ThT can bind parallel to theprotofilaments, their orientation gets partially randomized whileforming higher order aggregates (fibrils). To confirm the orienta-tion of ThT, we calculated the angle where r becomes maximum.We have found that r is maximum where the ThT orientation isalmost parallel to the fibril axis (Fig. S14b, ESI†), which is thenet direction of the emission dipoles in the fibrils similar topreviously reported results.42

Although it has been reported that selenium plays animportant role in intermolecular interactions, it is still not very clearwhat is the exact role of selenium in the amyloid formation.34,35 Weare in the process of understanding its role by changing the numberand nature of Sec residues in peptides.

Fig. 2 (a) FT-IR spectrum, (b) CD spectrum and (c) ThT fluorescence spectrum with (red) and without (cyan) peptide 1 in acetonitrile solution.

Fig. 3 Amyloid fibril formation via self-assembly of penta-selenopeptidesin acetonitrile. (a) Fluorescence image of spatially separated amyloid fibrilsspin-cast on glass coverslips. (b) High resolution TEM image showing discreteand higher order aggregates of amyloid fibrils. (c) AFM image of the penta-selenopeptide dropcast on mica. (d) XRD fiber diffraction pattern. (e) SEMimage showing the helical pitch length. (f) Maximum anisotropy image asobtained by wide field polarization resolved microscopy.

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To conclude, the present work demonstrates the synthesis of apenta-selenopeptide by SPPS using Sec-(Bzl)-OH as a precursor. Incontrast to the published literature where Sec-(Bzl)-OH has beenreported to have many limitations, this report demonstrates theuse of benzyl protected Sec for the synthesis of selenopeptides. Inaddition, we find that these peptides have a b-sheet conformationin acetonitrile and have a tendency to self-assemble into amyloidfibrils. Moreover, our work promises to stimulate the Sec-(Bzl)-OHbased SPSS to modulate the morphology of self-assembledselenopeptides in the future.

H. B. S. gratefully acknowledges the Department of Scienceand Technology, New Delhi, for a J. C. Bose Fellowship. A. C.acknowledges IRCC IIT Bombay and AFM central facilities.R. P. G. thanks UGC New Delhi and the Department of Chemistryfor allowing us to use central facility equipment. J. M. thanks IITBombay for fellowships and Ishan Rathore (BSBE) for valuablediscussions. The authors thank SAIF Bombay for SEM, TEM andprotein crystallography central facility at IIT Bombay.

Conflicts of interest

There are no conflicts to declare.

Notes and references1 L. Flohe, W. A. Gunzler and H. H. Schock, FEBS Lett., 1973, 32, 132–134.2 J. R. Arthur and G. J. Beckett, Br. Med. Bull., 1999, 55, 658–668.3 E. S. J. Arner and A. Holmgren, Eur. J. Biochem., 2000, 267, 6102–6109.4 J. C. Boyington, V. N. Gladyshev, S. V. Khangulov, T. C. Stadtman

and P. D. Sun, Science, 1997, 275, 1305–1308.5 I. M. Bell, M. L. Fisher, Z. P. Wu and D. Hilvert, Biochemistry, 1993,

32, 3754–3762.6 J. E. Cone, R. M. Del Rio, J. N. Davis and T. C. Stadtman, Proc. Natl.

Acad. Sci. U. S. A., 1976, 73, 2659–2663.7 K. Rajarathnam, B. D. Sykes, B. Dewald, M. Baggiolini and I. Clark-

Lewis, Biochemistry, 1999, 38, 7653–7658.8 M. Muttenthaler, S. T. Nevin, A. A. Grishin, S. T. Ngo, P. T. Choy,

N. L. Daly, S.-H. Hu, C. J. Armishaw, C.-I. A. Wang, R. J. Lewis,J. L. Martin, P. G. Noakes, D. J. Craik, D. J. Adams and P. F. Alewood,J. Am. Chem. Soc., 2010, 132, 3514–3522.

9 C. J. Armishaw, N. L. Daly, S. T. Nevin, D. J. Adams, D. J. Craik andP. F. Alewood, J. Biol. Chem., 2006, 281, 14136–14143.

10 S. Pegoraro, S. Fiori, S. Rudolph-Bohner, T. X. Watanabe andL. Moroder, J. Mol. Biol., 1998, 284, 779–792.

11 M. R. Sawaya, S. Sambashivan, R. Nelson, M. I. Ivanova, S. A. Sievers,M. I. Apostol, M. J. Thompson, M. Balbirnie, J. J. W. Wiltzius,H. T. McFarlane, A. O. Madsen, C. Riekel and D. Eisenberg, Nature,2007, 447, 453–457.

12 M. Mobli, D. Morgenstern, G. F. King, P. F. Alewood andM. Muttenthaler, Angew. Chem., Int. Ed., 2011, 50, 11952–11955.

13 A. D. Araujode, M. Mobli, G. F. King and P. F. Alewood, Angew.Chem., Int. Ed., 2011, 51, 10298–10302.

14 S. Shimodaira, Y. Asano, K. Arai and M. Iwaoka, Biochemistry, 2017,56, 5644–5653.

15 D. J. Selkoe, Nature, 2003, 426, 900–904.16 I. W. Hamley, Angew. Chem., Int. Ed., 2007, 46, 8128–8147.17 R. S. Jacob, D. Ghosh, P. K. Singh, S. K. Basu, N. N. Jha, S. Das,

P. K. Sukul, S. Patil, S. Sathaye, A. Kumar, A. Chowdhury, S. Malik,S. Sen and S. K. Maji, Biomaterials, 2015, 54, 97–105.

18 M. P. Mattson, Nature, 2004, 430, 631–639.19 F. Chiti and C. M. Dobson, Annu. Rev. Biochem., 2006, 75, 333–366.20 X. Du, J. Zhou, J. Shi and B. Xu, Chem. Rev., 2015, 115, 13165–13307.21 L. C. Serpell, Biochim. Biophys. Acta, 2000, 1502, 16–30.22 K. Tao, A. Levin, L. Adler-Abramovichab and E. Gazit, Chem. Soc.

Rev., 2016, 45, 3935–3953.23 X. Zhao and S. Zhang, Chem. Soc. Rev., 2006, 35, 1105–1110.24 M. Melchionna, K. E. Styan and S. Marchesan, Curr. Top. Med.

Chem., 2016, 16, 2009–2018.25 D. M. Ryan, S. B. Anderson, F. T. Senguen, R. E. Youngman and

B. L. Nilsson, Soft Matter, 2010, 6, 475–479.26 A. Bertolani, L. Pirrie, L. Stefan, N. Houbenov, J. S. Haataja,

L. Catalano, G. Terraneo, G. Giancane, L. Valli, R. Milani,O. Ikkala, G. Resnati and P. Metrangolo, Nat. Commun., 2015, 6,7574–7582.

27 L. Adler-Abramovich and E. Gazit, Chem. Soc. Rev., 2014, 43, 6881.28 M. L. Waters, Curr. Opin. Chem. Biol., 2002, 6, 736–741.29 E. Gazit, FASEB J., 2002, 16, 77–83.30 S. Xiong, W. R. Markesbery, C. Changxing and M. A. Lovell, Antioxid.

Redox Signaling, 2007, 9, 457–467.31 Z. Zhang, Q. Wu, C. Chen, R. Zheng, Y. Chen, Q. Liu, J. Ni and

G. Song, J. Alzheimer’s Dis., 2017, 59, 591–602.32 M. Muttenthaler and P. F. Alewood, J. Pept. Sci., 2008, 14, 1223–1239.33 T. Suzuki, H. Fujji, Y. Yamashita, C. Kabuto, S. Tanaka,

M. Harasawa, T. Mukai and T. Miyashi, J. Am. Chem. Soc., 1992,114, 3034–3043.

34 F. T. Burling and B. M. Goldstein, J. Am. Chem. Soc., 1992, 114,2313–2320.

35 A. K. Baev, Specific Intermolecular Interactions of Element-OrganicCompounds, Springer, 2015, p. 31.

36 Z. Arnon, L. Adler-Abramovich, A. Levin and E. Gazit, Isr. J. Chem.,2015, 55, 756–762.

37 D. Hall, R. Zhao, I. Dehlsen, N. Bloomfield, S. R. Williams,F. Arisaka, Y. Goto and J. A. Carver, Anal. Biochem., 2016, 498, 78–94.

38 M. Biancalana and S. Koide, Biochim. Biophys. Acta, Proteins Proteo-mics, 2010, 1804, 1405–1412.

39 J. C. Stroud, C. Liu, P. K. Teng and D. Eisenberg, Proc. Natl. Acad. Sci.U. S. A., 2012, 109, 7717–7722.

40 I. Maezawa, H. S. Hong, R. Liu, C. Y. Wu, R. H. Cheng, M. P. Kung,H. F. Kung, K. S. Lam, S. Oddo and F. M. L. Ferla, L. W. J. Neurochem.,2008, 104, 457–468.

41 S. Bhattacharya, D. K. Sharma, S. De, J. Mahato and A. Chowdhury,J. Phys. Chem. B, 2016, 120, 12404–12415.

42 J. Duboisset, P. Ferrand, W. He, X. Wang, H. Rigneault and S. Brasselet,J. Phys. Chem. B, 2013, 117, 784–788.

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