Modulating Kinetics of Amyloid-Like Aggregation of S. Aureus Phenol Soluble Modulins by Changes in Ph

Supplementary figures

Modulating kinetics of amyloid-like aggregation of S. aureus phenol soluble modulins by changes in pH

Masihuz Zaman and Maria Andreasen

1200 PSMα3

1000 pH 1 pH 2 800 pH 3 pH 4 600 pH 5 pH 6 pH 9 400

200 Fluorescence intensity (a.u.) intensity Fluorescence

0 0 10 20 30 40 Time (h) Figure S1: Aggregation of PSMα3 at 37 °C. Experimental kinetics of PSMα3 aggregation at 37°C every 10 min under quiescent conditions for single monomeric concentration (0.5 mg/ml) at pH 1-7.

a PSMα1 40 b PSMβ1 96

35 84

(h) 72

(h)

1/2

t 1/2 25 t 60

20 y = 53,382 + 0,19166x R= 0,94635 y = 16,668 + 0,054899x R= 0,95576 48

15 75 150 225 300 0 50 100 150 200 PSM1 (m) PSM1 (m) c 50 PSMβ2 d 80 δ-toxin

40 70

35 (hr)

(hr) 30 60

1/2

1/2 t 25

20 50 y = 43,888 + 0,087864x R= 0,94495 15 y = 46,647 - 0,15849x R= 0,97452

10 40 40 80 120 160 50 100 150 200 250 300 PSM2 (M) -toxin (m)

Figure S2: Half-time plots of PSMs. Dependence of t1/2 values on the various concentrations of PSMs peptides used in the aggregation assays. Four different concentrations for each peptide were studied at fixed pH. In all figures, the error bars correspond to the standard deviations of kinetic parameters determined for a single experiment in which each peptide concentration was run in triplicates. (a) Dependence of the t1/2 on the concentration of PSMα1. (b) Dependence of the t1/2 on the concentration of PSMβ1. (c) Dependence of the t1/2 on the concentration of PSMβ2. (d)

Dependence of the t1/2 on the concentration of δ-toxin.

a 8000 b 1,2 (A) PSM2 pH 2 -1 (B) PSM2 pH 10 6000 1

residue 4000

-1 0,8

2000 dmol

2 0,6 0 0,4 -2000

0,2 Normalized absorbnaceNormalized (a.u.)

MRE deg cm deg MRE -4000

-6000 0 190 200 210 220 230 240 250 1700 1680 1660 1640 1620 1600 -1 Wavelength (nm) Wavenumber (cm )

1 µM 2 µM

Figure S3: Structural and morphological characteristics of PSMα2 at 37°C. (a) Far-UV CD spectra of PSMα2 at pH 2 and pH 10. (b) Attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy of the amide I’ region (1600-1700 cm-1) of fibrils of PSMα2 at pH 10. (c) Transmission electron microscopy of PFSMα2 fibril at acidic (pH2) conditions. (d) Transmission electron microscopy of PSMα2 fibril at basic (pH10) condition.

a b 1,5 80 pH 2 PSMa2

PSMa3 ) PSMb2 -1 PSM1 1 60 PSM2 PSM3

residue 40 PSM4 0,5 -1 PSM1

20 PSM2 dmol 2 -toxin 0

0 Normalized ellipticity Normalized -0,5

-20 MRE (deg cm (deg MRE -40 -1 190 200 210 220 230 240 250 200 210 220 230 240 250 Wavelength (nm) Wavelength (nm) Figure S4: Far UV-CD structural characterization of PSM peptides. (a) Far UV-CD spectra of monomeric PSM peptides. (b) Far UV-CD spectra of PSMα2, PSMα3 and PSMβ2 at pH 2.

a 600 b 1000 (A) pH2 PSM1 PSM3 (B) pH10 PSM1 500 PSM4 PSM3 PSM1 PSM4 PSM2 800 PSM1 -toxin PSM2 400 -toxin 600 300 400

200

ANS F.I. (a.u.) F.I. ANS ANS F.I (a.u.) F.I ANS

100 200

0 0 400 450 500 550 600 400 450 500 550 600 Wavelength (nm) Wavelength (nm) Figure S5: ANS fluorescence emission spectra of PSMs fibrils. (a) ANS fluorescence emission spectra of PSMs fibrils at acidic (pH2) conditions. (b) ANS fluorescence emission spectra of PSMs fibrils basic (pH10) conditions.

Table S1: Charge and pKa of PSM peptides. Charge and pKa value of different peptides at numerous pH calculated by protein calculator v3.4.

Charge on Peptides pH PSMα1 PSMα2 PSMα3 PSMα4 PSMβ1 PSMβ2 δ-toxin pKa= 9.72 pKa= 10.00 pKa= 9.53 pKa= 9.72 pKa= 4.89 pKa= 5.69 pKa= 8.69

1.00 4.0 5.0 5.0 4.0 4.0 4.0 5.0 2.00 3.9 4.9 4.9 3.9 3.9 3.9 4.9 3.00 3.5 4.5 4.5 3.5 3.4 3.4 4.4 4.00 2.8 3.8 3.5 2.8 2.0 2.3 3.3 5.00 2.2 3.2 2.4 2.2 -0.2 0.6 1.6 6.00 2.0 3.0 2.0 2.0 -0.9 -0.2 1.1 7.00 1.9 2.9 1.9 1.9 -1.1 -0.8 0.9 8.00 1.5 2.5 1.5 1.5 -1.5 -1.5 0.5 9.00 0.8 1.7 0.7 0.8 -2.2 -2.1 -0.3 10.00 -0.5 0.0 -1.0 -0.5 -3.5 -3.0 -2.0 11.00 -1.7 -1.6 -2.6 -1.7 -4.7 -3.8 -3.6 12.00 -2.0 -2.0 -3.0 -2.0 -5.0 -4.0 -4.0

Table S2: Kinetic parameters of PSM aggregation. Summary of the averages and standard deviations of the kinetic parameters obtained from the ThT assays for different PSMs at various pH Values.

Peptide Peptide concentration t1/2 (hr) lag time (hr) (mg/ml) 0.25 mg/ml 21.8 ± 0.60 17.9 ± 0.62 PSMα1 (pH2) 0.37 mg/ml 26.4 ± 1.02 18.3 ± 0.69 0.50 mg/ml 30.1 ± 1.98 18.9 ± 0.30 0.62 mg/ml 30.7 ± 1.34 20.8 ± 1.93 0.25 mg/ml 61.8 ± 0.60 52.8 ± 0.72 PSMβ1 (pH10) 0.37 mg/ml 72.0 ± 10.0 53.1 ± 8.39 0.50 mg/ml 74.0 ± 1.73 44.7 ± 0.22 0.62 mg/ml 80.0 ± 8.25 42.0 ± 0.65 0.25 mg/ml 39.0 ± 1.08 34.5 ± 0.71 PSMβ2 (pH10) 0.37 mg/ml 31.5 ± 0.62 27.5 ± 0.74 0.50 mg/ml 28.5 ± 0.40 22.4 ± 0.43 0.62 mg/ml 25.3 ± 0.50 17.9 ± 0.86 0.30 mg/ml 51.0 ± 0.20 35.2 ± 0.32 δ-toxin (pH2) 0.45 mg/ml 60.0 ± 1.27 42.6 ± 0.77 0.60 mg/ml 62.6 ± 1.34 46.5 ± 0.26 0.75 mg/ml 65.3 ± 1.87 46.9 ± 1.09

Table S3: FTIR spectral deconvolution. The percentage contribution of various structural components is given for the aggregated form of all seven peptides at acidic (pH2) and basic (pH10) conditions.

pH 2 pH 10 Peptide Peak % β- % β- %-α % r- Peak % β- % β- %-α % r- position sheet turn helix coil position sheet turn helix coil PSMα1 1625, 1661 71.6 - 28.6 - 1626,1653, 47.2 9.0 29.4 - 1671 PSMα2 - - - - - 1604,1628, 2.67 1.45 - 40.5 1644,1660 PSMα3 1638,1656 36.1 - 34.8 - 1654 - - 81.1 - PSMα4 1628,1655, 56.7 19.6 23.7 - 1626, 1648 35.1 - - 43.2 1695 PSMβ1 1624,1664 51.1 29.5 - - 1629,1648, 46.1 17.0 - 11.2 1666 PSMβ2 - - - - - 1625,1645, 42.2 17.1 - 14.6 1665 δ-toxin 1625,1669 79.9 20.1 - - 1628,1645 3.2 - - 39.6