Supporting Information. Kinetic and Conformational Insights into Islet Amyloid Polypeptide Self-Assembly using a Biarsenical Fluorogenic Probe

Transcription

1 Kinetic and Conformational Insights into Islet Amyloid Polypeptide Self-Assembly using a Biarsenical Fluorogenic Probe Noé Quittot, Mathew Sebastiao, Soultan Al-Halifa and Steve Bourgault* Department of Chemistry, University of Québec in Montreal, Montreal, C.P. 8888, Succursale Centre- Ville, Montreal, H3C 3P8, Canada Quebec Network for Research on Protein Function, Engineering, and Applications, PROTEO * To whom correspondence should be addressed. bourgault.steve@uqam.ca

2 TABLE OF CONTENTS Figure S Figure S Figure S Figure S4. 6 Figure S Figure S Figure S Figure S Figure S Figure S Figure S Figure S Figure S Figure S Figure S Figure S

3 Figure S1. SDS-PAGE of photochemically cross-linked IAPP prefibrillar species. IAPP was incubated under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) at 50 μm for 30 min before PICUP treatment. 1: Molecular weight marker (MM). 2: IAPP prefibrillar species with PICUP treatment. 3: IAPP prefibrillar species without cross linking. 3

4 A) B) Figure S2. Dynamic light scattering analysis of IAPP prefibrillar species and amyloid fibrils. IAPP was solubilized in 20 mm Tris-HCl (ph 7.4) at 50 μm immediately followed by a filtration through a 0.22 μm polyvinylidene fluoride (PVDF) filter. The peptide was incubated for 30 min (prefibrillar species) and 24 h (fibrils). A) Correlation. B) Intensity distribution. 4

5 Figure S3. IAPP and S2S7 derivative were incubated under quiescent conditions at 25 C in 20 mm Tris-HCl buffer, ph 7.4, at 50 μm in presence of TCEP (500 μm) and BAL (125 μm). ThT (10 μm) was added after 30 min or 24 h of incubation, referred as prefibrillar species and amyloid fibrils respectively. ThT emission fluorescence spectra were recorded after excitation at 440 nm. 5

6 A) B) C) Figure S4. A, B) IAPP was incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris- HCl buffer (ph 7.4) supplemented with A) 500 μm TCEP, B) 500 μm TCEP and 70% hexafluoroisopropanol (HFIP). The fluorescence of FlAsH (0.5 μm) was measured every 10 min with excitation at 508 nm and emission at 533 nm. C) WCGGPCK was incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) supplemented with 0.5 μm FlAsH, 500 μm TCEP and 125 μm BAL, if indicated. Fluorescence was measured every 10 min with excitation at 508 nm and emission at 533 nm. 6

7 A) B) IAPP + FlAsH S2S7 + FlAsH C) D) Figure S5. A) IAPP and B) S2S7 were incubated under quiescent conditions at 25 C in 20 mm Tris- HCl buffer (ph 7.4) at 50 μm in presence of 0.5 μm FlAsH prior TEM analysis. Scale bar is 100 nm. C, D) Kinetics of self-assembly of IAPP (C) and S2S7 (D) monitored by ThT fluorescence. Peptides were incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) supplemented with 0.5 μm FlAsH, 500 μm TCEP and 125 μm BAL. Fluorescence of ThT (10 μm) was measured every 10 min with excitation at 440 nm and emission at 485 nm. 7

8 IAPP ThT FlAsH FRET Time (h) Figure S6. Kinetics of IAPP self-assembly monitored by ThT, FlAsH and ThT-to-FlAsH Förster resonance energy transfer (FRET). IAPP was incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) in presence of 10 μm ThT and/or 0.5μM FlAsH supplemented with 500 M TCEP and 125 M BAL. ThT, FlAsH or ThT-to-FlAsH fluorescence was measured every 10 min. 8

9 A) B) Figure S7. Kinetics of A) IAPP and B) S2S7 self-assembly in presence or absence of BAL and/or TCEP monitored by ThT fluorescence. Peptides were incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) supplemented with 500 M TCEP and/or 125 M BAL as indicated. Fluorescence of ThT (10 μm) was measured every 10 min with excitation at 440 nm and emission at 485 nm. 9

10 A) B) C) D) Figure S8. Dynamic light scattering analysis. CC-IAPP and IAPP-CC were solubilized in 20 mm Tris- HCl (ph 7.4) at 50 μm followed by a filtration through a 0.22 μm polyvinylidene fluoride (PVDF) filter. CC-IAPP and IAPP-CC were incubated for 30 min (prefibrillar species) and 24 h (amyloid fibrils) before DLS measurements. A, B) Correlation. C, D) Intensity distribution. 10

11 Figure S9. SDS-PAGE of CC-IAPP and IAPP-CC prefibrillar species photochemically cross-linked. Both analogs were incubated under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) at 50 μm for 30 min prior to cross-linking. 1: Molecular weight marker (MM: kdaltons). 2: CC-IAPP cross-linked prefibrillar species. 3: IAPP-CC cross-linked prefibrillar species. 11

12 A) B) C) D) IAPP-CC Fibrils Prefibrillar species [nm] Figure S10. Peptides were incubated under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) at a final concentration of 50 μm. A, B) Far-UV CD spectra were collected after 30 min (prefibrillar species) and 24 h (amyloid fibrils). C, D) ThT (10 μm), TCEP (500 M) and BAL (125 M) were added after 30 min or 24 h of incubation. Emission fluorescence spectra were recorded after an excitation at 440 nm. 12

13 A) B) Figure S11. Kinetics of A) CC-IAPP and B) IAPP-CC self-assembly in presence or absence of BAL and/or TCEP monitored by ThT fluorescence. Peptides were incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) supplemented with 500 M TCEP and/or M BAL, as indicated. ThT fluorescence of was measured every 10 min with excitation at 440 nm and emission at 485 nm. 13

14 A) B) CC-IAPP + FlAsH IAPP-CC + FlAsH C) D) Figure S12. A) CC-IAPP and B) IAPP-CC were incubated under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) at 50 μm in presence of 0.5 μm FlAsH prior to TEM analysis. Scale bar is 100 nm. C, D) Kinetics of CC-IAPP (C) and IAPP-CC (D) self-assembly monitored by ThT fluorescence. Peptides were incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris- HCl buffer (ph 7.4) supplemented with 0.5 μm FlAsH, 500 M TCEP and 125 M BAL. Fluorescence of ThT (10 μm) was measured every 10 min with excitation at 440 nm and emission at 485 nm. 14

15 A) B) Figure S13. Competition assay for FlAsH binding. A) WCGGPCK was incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) supplemented with 0.5 μm FlAsH, 500 μm TCEP and in presence of 125 μm of BAL, if indicated. Fluorescence was measured every 10 min with excitation at 508 nm and emission at 533 nm. B) WCGGPCK was incubated at 12.5 μm under quiescent conditions at 25 C for 1 h in 20 mm Tris-HCl buffer (ph 7.4) supplemented with 0.5 μm FlAsH, 500 μm TCEP and increasing BAL concentrations. Emission fluorescence spectra from 520 to 600 nm were recorded after an excitation at 508 nm. The maximum emission at 533 nm was used to determine the relative fluorescence, i.e. with a ratio between each spectrum and fluorescence intensity in presence of the lowest BAL concentration. 15

16 A) B) C) Figure S14. Dynamic light scattering analysis. riapp-cc was solubilized in 20 mm Tris-HCl (ph 7.4) at 50 μm followed by a filtration through a 0.22 μm polyvinylidene fluoride (PVDF) filter. riapp-cc was incubated for 24 h preceding measurements. A) Number distribution. B) Intensity distribution. C) Correlation. 16

17 A) B) C) Figure S15. A) riapp, B) CC-rIAPP and C) riapp-cc were incubated at 12.5 μm under quiescent conditions at 25 C in 20 mm Tris-HCl buffer (ph 7.4) supplemented with 0.5 M FlAsH, 500 μm TCEP and 125 μm BAL, if indicated. Fluorescence was measured every 10 min with excitation at 508 nm and emission at 533 nm. 17

18 CC-pfIAPP Prefibrillar species [nm] Fibrils Figure S16. CC-pfIAPP was incubated under quiescent conditions at 25 C in 20 mm Tris-HCl, ph 7.4 at a concentration of 50 μm. Far-UV CD spectra were recorded after 30 min (prefibrillar species) and 24 h (fibrils) incubation. 18