Articleshttps://doi.org/10.1038/s41563-019-0343-2

Rigid helical-like assemblies from a self-aggregating tripeptideSantu Bera   1, Sudipta Mondal1, Bin Xue2, Linda J. W. Shimon   3, Yi Cao   2 and Ehud Gazit1*

1Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel. 2Collaborative Innovation Center of Advanced Microstructures, National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University, Nanjing, Jiangsu, China. 3Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel. *e-mail: ehudg@post.tau.ac.il

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Supplementary Figure 1: AFM images of Pro-Phe-Phe fibers. a-d, AFM images (a, c) and the

corresponding height profile along the red lines (b, d) of two different single fibers. The height

profile of the fibers exhibit nearly identical spatial fluctuation along the fiber axis at a regular

interval, confirming their helical nature.

3

Supplementary Figure 2: Kinetics of Pro-Phe-Phe self-assembly. HR-SEM images of Pro-Phe-

Phe in phosphate buffer at pH 7.4 over time. The peptide transformed from unstructured to self-

assembled twisted fiber morphology within 15 days. Scale bars, 10 µm.

Supplementary Figure 3: Critical aggregation concentration of Pro-Phe-Phe. a, UV-visible

spectra of the tripeptide at two different concentrations, 0.1 mg/ml (black) and 7 mg/ml (red). b,

4

Plot of the optical density versus log[Pro-Phe-Phe]. From the fitting curve, the critical aggregation

concentration was found to be ~1.2 mg/ml.

Supplementary Figure 4: ThT binding of Pro-Phe-Phe. a, Confocal fluorescence microscopy

image of 20 mM tripeptide fibers stained with 20 µM ThT in phosphate buffer at pH 7.4. b, ThT

fluorescence emission spectrum of Pro-Phe-Phe fibers upon excitation at 440 nm. Pre-assembled

peptide samples at a concentration of 20 mM, or phosphate buffer as a control, were added at 1:1

volume to 40 µM ThT solution in phosphate buffer. c, ThT kinetic experiment showing

fluorescence emission intensity at 485 nm for 20 µM ThT incubated with and without 20 mM

peptide, as recorded over 12h examined with the preformed matured fibers. Standard errors are

indicated by error bars. d, Confocal fluorescence microscopy images of Pro-Phe-Phe fibers upon

completion of kinetic measurements. Scale bars of a and d, 10 µm. Result of c is presented as

mean ± s.d. of three independent experiments.

5

Supplementary Figure 5. CD spectra of Pro-Phe-Phe. The spectra were acquired for 5 mg/ml

peptide solution in phosphate buffer. The tripeptide adopted a helical conformation, which was

stable at a wide range of temperatures, as indicated. Upon temperature increase, the peptide

underwent partial unfolding, as evident from the decrease in molar ellipticity at 210 nm.

200 220 240

-1.5

-1.0

-0.5

0.0

0.5

1.0

Elip

tic

ity

(md

eg

)

Wavelength (nm)

250C

500C

700C

900C

6

Supplementary Figure 6: Fiber diffraction of Pro-Phe-Phe. a, The PXRD diffractogram of the

Pro-Phe-Phe fiber. b, Graph of whole profile fitting on Pro-Phe-Phe diffractogram. The observed

and calculated diffractograms are represented as green line and blue dots, respectively. The

difference between them (Io − Ic) is shown in a cyan line and the background is marked by a black

line. c, Comparison of X-ray diffraction of fiber (black) and simulated powder pattern obtained

from single crystal data (red). Both diffractions show a similar peak pattern with unaltered position

of most of peaks (marked in blue), signifying a similar molecular arrangement in both states. The

wide-angle peak at 5.46 Å (marked in yellow) in the crystal is slightly shifted to 5.27 Å in the

fibers, indicating the latter to comprise a stronger interaction between helical-like arrangements

along the long axis.

7

Supplementary Figure 7: Crystal structure of Pro-Phe-Phe. a, Surfaces of a single helix are

shown by 90° rotations. Figures show two faces of the helix are hydrophobic in nature by exposing

aromatic ring of Phe residues. The other two faces are hydrophilic in nature by extending the amide

groups. b, Crystal packing of Pro-Phe-Phe down the elongated axis (left) shows stacking of helical

sheets through hydrophobic interaction. Rotation of 90° (right) shows hydrophilic interaction

among the helical stands inside individual sheet1,2. c, Unit cell measurement of the crystal with

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respect to crystal morphology. Single crystal is shown mounted on a MiTeGen loop. The crystal is

highlighted in white box and the respective cell axes are shown in red. The morphological long axis

of the crystal is aligned along the crystallographic a axis of the unit cell. Size of the crystal is 0.27

x 0.10 x 0.02 mm.

Supplementary Figure 8: Crystal structure of Hyp-Phe-Phe. a, ORTEP diagram of the

asymmetric unit in 50% probability displacement ellipsoids. The asymmetric unit of Hyp-Phe-Phe

was comprised of two molecules and shared a common H-bonding pattern. b, Single helix in the

crystallographic b-direction with its dots represented by colors according to hydrophobicity, as

indicated. c, Aromatic zipper-like structural organization of Hyp-Phe-Phe in the bc plane.

9

Supplementary Figure 9: Schematic model showing a fibrillary assembly composed of super-

helical units. Scale bar, 5 µm.

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5

0

50

100

150

200

Fre

qu

en

cy

Log10(Youngs Modulus)

47 GPa

Supplementary Figure 10: Mechanical strength of Pro-Phe-Phe self-assembled fibers. The

Young’s modulus values obtained from fibers were mostly in the same range as that of crystals,

indicating a similar molecular arrangement and robustness in both states.

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Supplementary Note 1:

Assemblies, structures of Ala-Phe-Phe and Ala-Phe-Ala

The modified tripeptide Ala-Phe-Phe self-assembled in phosphate buffer at pH 7.4,

producing flake-like morphologies completely different from the helical fibers of Pro-Phe-

Phe (Supplementary Figure 11a). The secondary conformation of the Ala-Phe-Phe

assembled structures was analyzed by FTIR and CD. The FTIR spectrum showed a sharp

amide I band at 1637 cm-1, instead of 1647 cm-1 as observed for Pro-Phe-Phe, indicating

the presence of a β-sheet structure (Supplementary Figure 11b)3,4. The CD spectrum of the

Ala-Phe-Phe self-assemblies showed positive peaks near 220 nm due to the presence of

aromatic residues (Supplementary Figure 11c)5,6. The conformation and self-assembly at

the atomic level was further confirmed by X-ray crystallography of the tripeptide. Single

crystals suitable for X-ray diffraction were obtained by slow evaporation of the methanol-

water solution. Two tripeptide molecules were crystalized with one molecule of

trifluoroacetic acid and one water molecule in the asymmetric unit in P1 space group, as

shown in 50% probability displacement ellipsoids (Supplementary Figure 11d). No

intramolecular H-bond was detected. The two Phe side chains were arranged in opposite

direction with respect to the peptide backbone. The torsion angles around the Phe2 residue

appeared to play a pivotal role in determining the overall structural features. The allowed

torsion angles of the Phe2 residue were constrained within the β-sheet region of the

Ramachandran plot, with φ2 and ψ2 values of -144.20°, 150.81° for molecule A and -

127.95°, 135.10° for molecule B, respectively (Supplementary Figure 11e). In the

crystallographic a-direction, β-strands were interacted with the adjacent strands through

intermolecular H-bonds and stacked in antiparallel manner, thereby producing an

antiparallel β-sheet conformation (Supplementary Figure 11f). Adjacent β-sheets were

11

Supplementary Figure 11: Self-assembly of Ala-Phe-Phe. a, HR-SEM micrographs of the

tripeptide flakes prepared in phosphate buffer at pH 7.4. Scale bar, 20 µm. b, FTIR analysis of the

tripeptide. c, CD spectrum of the tripeptide in solution. d-g, Single crystal structure, d, ORTEP

diagram of the asymmetric unit in 50% probability displacement ellipsoids, e, β-strand, f,

Antiparallel β-sheet structure of the tripeptide through intermolecular N-H…O hydrogen bonds

along the a-direction, g, Cross-β structure.

12

stabilized through π-π interactions of aromatic residues and produced an aromatic zipper-

like structure (Supplementary Figure 11g). Thus, Ala-Phe-Phe overall arrangement is a

cross-β-like structure, a fundamental secondary structural module previously/frequently

observed in self-assembled nanostructures formed by short peptide sequences7-11. Hence,

modification of Pro-Phe-Phe to Ala-Phe-Phe changes the secondary structure of the

tripeptide from helical-like to β-sheet, clearly demonstrating the generic nature of the

backbone.

Further modification of the backbone was performed by replacing terminal Phe residue

with Ala. The modified tripeptide, Ala-Phe-Ala, failed to show any well-ordered

morphology in phosphate buffer at pH 7.4, as observed by HRSEM imaging

(Supplementary Figure 12a). The FTIR secondary conformation analysis showed a sharp

amide I band at 1631 cm-1 along with a shoulder at 1687 cm-1, indicating the predominant

presence of a β-sheet structure (Supplementary Figure 12b)3,4. Similar to Ala-Phe-Phe, CD

spectrum of the Ala-Phe-Ala tripeptide assemblies also showed positive peaks near 220

nm due to the presence of aromatic residues (Supplementary Figure 12c)5,6. Atomic level

conformation was investigated by single crystal structure analysis. Single crystals suitable

for X-ray diffraction were obtained by slow evaporation of 9:1 methanol/water solution.

The asymmetric unit was found to consist of two tripeptide molecules along with one

molecule of trifluoroacetic acid, acetic acid and water in the P21 space group shown in 50%

probability displacement ellipsoids (Supplementary Figure 12d). The two tripeptide

molecules adopted a similar backbone conformation, except for a slight variation in the

torsion angle. No intramolecular hydrogen bond was observed inside either of the two

molecules. The allowed torsion angles of the Phe2 residue were constrained within the β-

13

Supplementary Figure 12: Self-assembly of Ala-Phe-Ala. a, HR-SEM micrographs of the

tripeptide prepared in phosphate buffer at pH 7.4. Scale bar, 20 µm. b, FTIR analysis of the

tripeptide. c, CD spectrum of the tripeptide in solution. d-g, Single crystal structure, d, ORTEP

diagram of the asymmetric unit in 50% probability displacement ellipsoids, e, β-strand, f,

Antiparallel β-sheet structure of the tripeptide through intermolecular N-H…O hydrogen bonds

along the a-direction, g, Cross-β structure.

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sheet region of the Ramachandran plot, with φ2 and ψ2 values of -141.77°, 144.12° for

molecule A and -140.72°, 130.43° for molecule B, respectively (Supplementary Figure

12e). Previously reported conformational analysis of Ala-Phe-Ala based on both

experimental and molecular dynamics studies also predicted similar torsion angles for the

extended-β structure12-14. The tripeptide exhibited two conventional NH3+... –OOC head-

to-tail hydrogen bonds, with the third amine hydrogen involving the acetic acid or

trifluoroacetic acid moiety. In the crystallographic a-direction, the β-strands were stacked

in an antiparallel manner, interacting with the adjacent strands through intermolecular H-

bonds incorporating both terminal polar groups and internal amide groups, thereby

producing an antiparallel β-sheet conformation (Supplementary Figure 12f). Nearby sheets

were connected through head-to-tail H-bonds between polar head groups, generating a 1D

layer where all the Phe residues are positioned on the same side of the layer. The individual

layers stacked to afford a layer-by-layer structure of Ala-Phe-Ala stabilized through van

der Waal interactions. The overall molecular packing of Ala-Phe-Ala comprised of a cross-

β-like structure (Supplementary Figure 12g). Notably, no π-π interactions between Phe

residues were observed, neither within cross-strands along the a-direction nor within cross-

sheets along the b-direction. In conclusion, both modified tripeptides, Ala-Phe-Phe and

Ala-Phe-Ala, organized into a cross-β-like structure rather super-helix, signifying the

generic nature of Pro-Phe-Phe peptide backbone to arrange into a helical-like structure.

15

Supplementary Note 2:

Assemblies, structures of Phe-Pro-Phe and Phe-Phe-Pro

The generic nature of the helical-like conformation of Pro-Phe-Phe and its attribution to

the peptide backbone were further investigated by modifying the amino acid sequence of

Pro-Phe-Phe into either Phe-Pro-Phe or Phe-Phe-Pro. The self-assembled morphology and

secondary structures were studied by HRSEM, CD, FTIR and PXRD analysis. Phe-Pro-

Phe assembled in phosphate buffer at pH 7.4 to produce thin fibers along with spherical

morphologies (Supplementary Figure 13a). The length and width of the fibers were much

smaller compared to the Pro-Phe-Phe helical fibers. FTIR spectrum showed a sharp amide

I band at 1642 cm-1 indicating the predominate presence of a β-sheet structure

(Supplementary Figure 13b)15. CD analysis was employed to further confirm the secondary

structure. Phe-Pro-Phe self-assemblies showed positive peaks near 228 nm, along with a

negative maximum at 212 nm (Supplementary Figure 13c). The positive CD band near

225-235 nm generally arises from the contribution of aromatic residues5. Previous studies

on Phe-Phe–based nanostructures designated these patterns as β-turn structures. The

negative CD band indicates the presence of a typical β-sheet signature16. Thus, both the

FTIR and CD analyses clearly demonstrated the absence of a helical conformation for Phe-

Pro-Phe, in contrast to Pro-Phe-Phe. The X-ray diffraction of the dried assembled samples

showed a unit cell similar to Pro-Phe-Phe (Supplementary Figure 13d). The best match of

the powder pattern suggested monoclinic unit cells with a=5.316Å, b=36.530Å, c=10.777Å

and β=97.34° (Supplementary Figure 14). However, the secondary structure analysis

indicated the assembly of distinctive units compared to that of Pro-Phe-Phe.

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Supplementary Figure 13: Self-assembled structure of Phe-Pro-Phe and Phe-Phe-Pro. (a-d)

Phe-Pro-Phe. (e-h) Phe-Phe-Pro. a, e, HR-SEM micrograph in phosphate buffer at pH 7.4. Scale

bars, 500 nm. b, f, FTIR analysis. c, g, CD spectrum. d, h, X-ray diffraction pattern.

17

Supplementary Figure 14: Powder diffraction of Phe-Pro-Phe. Graph of whole profile fitting

on Phe-Pro-Phe diffractogram is shown. The observed and calculated diffractogram are represented

in green line and blue dots, respectively. The difference between them (Io − Ic) is shown in a cyan

line and the background is marked by a black line.

The other modified tripeptide, Phe-Phe-Pro, self-assembled into a spherical

nanostructure in phosphate buffer at pH 7.4 (Supplementary Figure 13e). The FTIR spectra

showed a sharp amide I band at 1628 cm-1 along with a peak at 1678 cm-1, indicating

predominately a β-sheet secondary structure (Supplementary Figure 13f)15. CD spectra also

presented a typical β-sheet like pattern, displaying a negative maximum at 211 nm along

with a positive peak at 200 nm (Supplementary Figure 13g)17. PXRD study of the dried

sample showed the presence of amorphous materials (Supplementary Figure 13h). Thus,

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both modified tripeptides, Phe-Pro-Phe and Phe-Phe-Pro, were found incapable of forming

a cross-helical structure, further confirming the helical assembly to be a truly generic nature

of the Pro-Phe-Phe peptide backbone.

19

Supplementary Figure 15: Characterization of Pro-Phe-Phe. a, Mass Spectra, b, HPLC trace.

20

Supplementary Figure 16: Characterization of Hyp-Phe-Phe. a, Mass Spectra, b, HPLC trace.

21

Supplementary Figure 17: Characterization of Ala-Phe-Phe. a, Mass Spectra, b, HPLC trace.

22

Supplementary Figure 18: Characterization of Ala-Phe-Ala. a, Mass Spectra, b, HPLC trace.

23

Supplementary Figure 19: Characterization of Phe-Pro-Phe. a, Mass Spectra, b, HPLC trace.

24

Supplementary Figure 20: Characterization of Phe-Phe-Pro. a, Mass Spectra, b, HPLC trace.

25

Supplementary Table 1: Comparison of Phe2 torsion angle of the studied tripeptides.

Sequence φ2 ψ2 Conformation

Pro-Phe-Phe

-78.5°

-38.9°

helical-like

Hyp-Phe-Phe

Molecule A

Molecule B

-71.1°

-70.5°

-43.2°

-41.9°

helical-like

Ala-Phe-Phe

Molecule A

Molecule B

-144.20°

-127.95°

150.81°

135.10°

β-sheet

Ala-Phe-Ala

Molecule A

Molecule B

-141.77°

-140.72°

144.12°

130.43°

β-sheet

26

Supplementary Table 2. List of peptides studied and their preferred molecular

conformations.

Sequence Conformation

Pro-Phe-Phe super-helical

Hyp-Phe-Phe super-helical

Ala-Phe-Phe β-sheet

Ala-Phe-Ala β-sheet

Phe-Pro-Phe ~ β-sheet

Phe-Phe-Pro β-sheet

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Supplementary Table 3: Data collection and refinement statistics

Experimental details:

Crystal data Pro-Phe-Phe Hyp-Phe-Phe

Chemical formula C23 H27 N3 O4 C23 H27 N3 O5

Mr 409.47 425.47

Crystal system Monoclinic triclinic

Space group P21 P1

a (Å) 5.3214(1) 5.4321(9)

b (Å) 11.5689(1) 11.891(1)

c (Å) 17.0398(1) 16.765(2)

α (°) 90 84.57(1)

(°) 97.2170(10) 83.00(1)

γ (°) 90

V (Å3) 1040.71(2) 1070.0(3)

Z , Z’ 2 2

(mm–1) 0.096 0.771

Temperature (K) 100 (2) 100 (2)

Data collection

Diffractometer Rigaku XtaLAB

Pro: Kappa dual

home/near

Rigaku XtaLAB

Pro: Kappa dual

home/near

Crystal size (mm) 0.27/0.10/0.20 0.262/0.090/0.016

Absorption

correction

multi-scan multi-scan

Tmin , Tmax 0.826, 0.985 0.684, 1.000

Nmeasured 17218 9432

Nobserved [I > 2(I)] 4446 4441

Rint 0.0373 0.0782

max (°) 79.898 52.852

Refinement

R[F2 > 2(F2)] 0.0383 0.0834

wR(F2) 0.0957 0.2247

Goodness-of-fit 1.105 1.040

No. of reflections 4446 4441

No. of parameters 271 561

No. of restraints 1 1

H-atom treatment H-atom parameters

constrained

H-atom parameters

constrained

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Supplementary Table 4: Data collection and refinement statistics

Experimental details:

Crystal data Ala-Phe-Phe Ala-Phe-Ala

CCDC

Diffractometer Rigaku XtaLabPro Rigaku XtaLabPro

Empirical formula 2(C21 H25 N3

O4),C2F3O2, O

2(C15 H21 N3 O4),

C2F3O2, C2H4O2, H2O

Crystal description 895.90 805.78

Formula weight (g/mol) Colourless wedge prism Colourless plate

Temperature (K) 100 100

Wavelength (Å) 1.54184 1.54184

Crystal system Triclinic Monoclinic

Space group P1 P21

a (Å) 9.49980(10) 9.5010(1)

b (Å) 10.9522(2) 17.5658(1)

c (Å) 11.9932(2) 12.1344(1)

() 111.088(2) 90

() 99.231(1) 99.183(1)

() 90.369(1) 90

Volume (Å3) 1146.38(3) 1999.19(3)

Z 1 4

Density calculated (Mg/m3) 1.298 1.339

Absorption coefficient (mm-1) 0.860 0.958

F(000) 471 850

Crystal size (mm3) 0.12x0.04x0.01 0.131x0.040x0.031

Theta range for data collection

()

4.68 to 79.410 4.468 to 80.206

Reflection collected (Unique) 31351(9083) 44321(8641)

R int 0.0397 0.0389

Completeness 96.9% 95.5

Data\restraints\parameters 9083/3/581 8641/4/521

Goodness-of-fit on F2 1.052 1.027

Final R [I>2(I)] R1=0.0505,

wR2=0.0819

R1=0.339 wR2=0.0863

R (all data) R1=0.0814 wR2=0.0908 R1=0.0345,

wR2=0.0867

Largest diff. peak and hole (e.

Å-3)

0.757 and -0.385 0.333 and -0.223

29

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