Supporting Information

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1 Supporting Information Inhibiting the Fibrillation of Serum Albumin Proteins in the Presence of Surface Active Ionic Liquids (SAILs) at Low ph: Spectroscopic and Microscopic Study Sangita Kundu, Chiranjib Banerjee and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur , WB, India Fax: Instrumentation: 1.1. ThT Fluorescence: The formation and inhibition of the BSA fibril are monitored by measuring ThT fluorescence intensity. After completion of incuabation period, the aliquots are taken from different sets of solutions in presence and absence of SAILs and mixed with ThT. The concentrations of fibril and ThT are maintained at 50 M and 3 M respectively. The fluorescence intensity of ThT is recorded in BSA fibril, to confirm the fibril formation. The effects of SAILs on BSA fibril are also monitored by observing the changes in fluorescence intensity of ThT. The following parameters are adjusted for monitoring ThT fluorescence intensity during aggregation experiments: λex = 450 nm, excitation bandpass = 5nm, and emission bandpass = 2.5 nm.the steady state fluorescence measurements are performed using a 1 cm quartz cell in Fluorolog Circular Dichroism (CD) Spectroscopy: The near-uv CD spectra were recorded at 298 K using a Jasco J-815 CD spectrometer. A quartz cuvette (path length 10 mm) was used for the measurements. The secondary structural changes, in the range of nm, were recorded using a scan rate of 100 nm/min; the final spectrum was averaged over three scans. Baseline subtraction with Milli Q water and respective SAILs were done to correct the obtained spectra Field Emission Scanning Electron Microscopy (FESEM): 1

2 Fl. Intensity (a.u.) For FESEM measurements, the sample cast film on an aluminum foil was first air-dried at room temperature and then stored in desiccators before measurement. A layer of gold was sputtered on top and the sample was examined on Supra 40 (Carl Zeiss Pvt. Ltd) instrument Atomic Force Microscopy (AFM): Atomic force microscopy (AFM) was performed on Agilent 5500 in the tapping mode. The sample was diluted to a BSA concentration of 0.3 µm and was placed on a newly cleaved mica surface, and the sample was spin-coated and air-dried overnight before imaging Surface tension: Surface tension measurements were carried out using the Du Nuo y ring detachment method with an automated surface tensiometer (3S, GBX, France). All the experiments were performed at a constant temperature of 30 C. For each concentration, three measurements for γ were performed, and their mean was taken as the value of the equilibrium surface tension. The surface tension of Milli-Q water was 72 (±0.1) mnm x x10 6 BSA berore fibrillation BSA fibril 1.0x x x x x Wavelength (nm) Figure S1: (a) Emission spectra of ThT in BSA before fibrillation and BSA fibril. 2

3 CD (mdeg) 50 0 native bsa bsa at 0 h bsa fibril Wavelength (nm) Figure S2: Circular Dichroism (CD) spectra of native bsa, bsa fibril at 0 hour and bsa fibril after 24 hours. (a) (b) (c) (d) Figure S3: (a),(b) Fluorescence intensity images of BSA fibril after 7 days and (c), (d)after 15 days. 3

4 Figure S4: AFM image of BSA fibril. (a) (b) (c) Figure S5: FESEM images of BSA fibril inhibition by (a) 5.8 mm C 8 mimcl (scale bar, 400 µm), (b) 0.29 mm C 12 mimcl (scale bar, 20 µm) and (c) 0.08 mm C 16 mimcl (scale bar, 50 µm). 4

5 Intensity (a.u.) (a) (b) (c) Figure S6: Fluorescence intensity images of BSA fibril inhibition by the addition of different SAILs, (a) 5.8 mm C 8 mimcl, (b) 0.29 mm C 12 mimcl, (c) 0.08 mm C 16 mimcl Lifetime (ps) Figure S7: Lifetime distribution histogram of DCM inside the BSA fibril. 5

6 G( ) Time (ns) Figure S8: Fluorescence correlation curves of DCM in BSA fibril considering single component diffusion model. (a) (b) Figure S9: (a) Fluorescence intensity and (b) lifetime images of HSA fibril. 6

7 (a) (b) (c) Figure S10: Fluorescence intensity images of HSA fibril in presence of (a) 5.8 mm C 8 mimcl, (b) 0.29 mm C 12 mimcl and (c) 0.08 mm C 16 mimcl. Figure S11: FESEM images of HSA fibrils (scale bar, 500 µm). (a) (b) (c) Figure S12: FESEM images of HSA fibrils after addition of (a) C 8 mimcl (scale bar, 400 µm), (b) C 12 mimcl (scale bar, 10 µm) and (c) C 16 mimcl (scale bar, 20 µm). 7

8 Fl. Intensity (a.u.) Fl. Intensity (a.u.) Fl. Intensity (a.u.) I1/I3 1.6 (a) IL concn. (mm) Increasing IL concentration I1/I3 1.6 (b) IL concn. (mm) Increasing IL concentration I1/I3 1.6 (c) IL concn. (mm) Increasing IL concentration Wavelength(nm) Wavelength(nm) Wavelength(nm) Figure S13: Fluorescence spectra of pyrene ((λ ex = 334 nm) of (a) C 8 mimcl/ H 2 O, (b) C 12 mimcl/ H 2 O, (c) C 16 mimcl/ H 2 O, whereas the corresponding I 1 /I 3 ratio (I 1 at λ em = 374 nm and I 3 at λ em = 384 nm) of pyrene are shown in the inset of the figures. Table S1. Determination of surface tension of different concentrations of SAILs. SAIL Concentration (mm) Surface Tension (mnm -1 ) C 8 mimcl ± ± 0.1 C 12 mimcl ± ± 0.1 C 16 mimcl ± ± 0.1 Complete author list for references 24, 31 (24) Pepys, M. B.; Hawkins, P. N.; Booth, D. R.; Vigushin, D. M.; Tennent, G. A.; Soutar, A. K.; Totty, N.; Nguyen, O.; Blake, C. C. F.; Terry, C. J.; Feest, T. G.; Zalin, A. M.; Hsuan, J. J. Human Lysozyme Gene Mutations Cause Hereditary Systemic Amyloidosis. Nature 1993, 362,

9 (31) Sinha, S.; Lopes, D. H. J.; Du, Z.; Pang, E. S.; Shanmugam, A.; Lomakin, A.; Talbiersky, P.; Tennstaedt, A.; McDaniel, K.; Bakshi, R.; Kuo, P.-Y.; Ehrmann, M.; Benedek, G. B.; Loo, J. A.; Klärner, F.-G.; Schrader, T.; Wang, C.; Bitan, G. Lysine-Specific Molecular Tweezers Are Broad-Spectrum Inhibitors of Assembly and Toxicity of Amyloid Proteins. J. Am. Chem. Soc. 2011, 133,