Supplementary Information

Supplementary Figure legends

Fig. S1. Tyr intrinsic fluorescence of physalaemin. Peptide at a concentration of 2 mg/ml was incubated in 5% D-mannitol, 0.01% sodium azide with slight agitation at 37°C in presence and absence of heparin. Intrinsic fluorescence was performed with excitation at 280 nm and emission in the range of 290-400 nm. A) Tyr fluorescence of physalaemin. B) Tyr fluorescence of physalaemin in presence of heparin. The fluorescence intensity was increased when peptide was incubated for 15 days in presence of heparin suggesting change in Tyr microenvironment during incubation. C) Stern-Volmer plot of Tyr quenching suggesting that Tyr of peptide incubated in presence of heparin is less solvent exposed compared to 15 days incubated sample in absence of heparin.

Fig. S2. Effect of TFE on the conformation and aggregation of physalaemin. A) Increasing percentage (v/v) of TFE was added to 70 mM solution of freshly prepared peptide in water. The CD spectra are shown in presence of 0 to 80% of TFE. Induction of helicity was observed in TFE concentration dependent manner. B) ThT binding study of the peptide in presence of 0-80% TFE. Physalaemin in presence of 20-50% TFE showed large amount of ThT binding indicating the formation of amyloids. C) CD spectra of one week incubated peptide sample in various concentration of TFE showed no significant conformation transition to b-sheet. D) AFM images of one week incubated sample in presence of 30% TFE suggesting amyloid-like fibril formation at room temperature. Left and right hand side images are height and amplitude images, respectively.

Fig. S1

Fig S2


Table S1: TANGO Vs WALTZ prediction for the peptides reported to form amyloid in vitro*

Peptide / Peptide Names / Peptide Sequences / TANGO / WALTZ / Amyloid (experiment)
1 / VIP / HSDAVFTDNYTRLRKQMA
VKKYLNSILN / - / + / +
2 / sCalcitonin / CSNLSTCVLGKLSQELHKL
QTYPRTNTGSGTP / - / + / +
3 / Obestatin / FNAPFDVGIKLSGVQYQQ
HS / - / + / +
4 / pNPY / YPSKPDNPGEDAPAEDLA
RYYSALRHYINLITRQRY / - / + / +
5 / h-GRF-40 / YADAIFTNSYRKVLGQLSA
RKLLQDIMSRQQGESNQERGA / - / + / +
6 / mUcnIII / FTLSLDVPTN IMNILFNIDK AKNLRAKAAA NAQLMAQI / - / + / +
7 / h-GRF-44 / YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL / - / + / +

*A test study of predicting amyloid propensities of 7 peptides reported to form amyloids in vitro by TANGO and WALTZ. The data suggest that although the experimental results are in consistent with WALTZ prediction (with high sensitivity), TANGO did not show any amyloid prone region in any of these peptides. These results point out the sensitivity differences of these two algorithms.


Table S2: Comparative prediction of amyloid propensities of Ab(25-35) and tachykinins*

Peptides / Amyloid Prediction Algorithm
TANGO / WALTZ
(high sensitivity) / PASTA / AGGRE SCAN
(hot spots) / FOLD AMYLOID / Amyl Pred / Net CSSP
Ab(25-35) / + / + / + / + / + / + / +
Physalaemin / - / + / + / - / - / - / +
Substance P / - / + / + / + / + / + / +
Kassinin / - / - / + / + / - / - / +

*Seven amyloid prediction algorithms TANGO, WALTZ, PASTA, AGGRESCAN, FOLDAMYLOID And NetCSSP were used to predict the amyloid prediction propensities of Ab(25-35) and tachykinin peptides. "+" sign indicates the positive amyloid propensity and "–" sign indicates no amyloid propensity. WALTZ was performed with high sensitivity. AGGRESCAN shows the presence and absence of aggregation hot spots. This comparative study suggests that all the tachykinins under study have some intrinsic amyloid formation tendency. However Ab(25-35) has significantly higher amyloid propensity compared to these neuropeptides.