1. Study of hIAPP folding with FTIR and 2DIR Yun L. Ling, Dave B. Strasfeld, Sang-Hee Shim, and Martin T. Zanni Department of Chemistry, University of Wisconsin-Madison hIAPP type 2 diabetes 50 um Fibril in pancreas

2. Motivation: More explicit insight of hIAPP folding There is limited structural information on intermediates Langen, R. et al., Biochemistry 2005, 44, 12113 It is not fibril but intermediate that causes pancreatic cell death. 1. FTIR and 2D IR are sensitive to peptide secondary structure 2. They have the time-resolution to catch intermediates Random coil Beta-sheet

3. Method: How FTIR is sensitive to secondary structure? 1645 cm -1 Frequency (cm -1 ) random coil Frequency (cm -1 ) alpha-helix beta-sheet 1635 cm -1 1685 cm -1 1610 cm -1

4. Result: FTIR spectrum of fully folded hIAPP It is more than beta-sheet. 1620 cm -1 (beta-sheet) 1645 cm -1 (random coil)

5. Nd:YVO 4 CW laser Ti:Sapphire Oscillator Stretcher Regenerative Amplifier Compressor Nd:YLF ns laser  -BBO based OPA Difference Frequency Mixing w/ AgGaS 2 800 nm, 88 MHz. 2 nJ/pulse idler Ge AOM Pulse Shaper Tunable mid-IR 3-10  m, 2-5  J/pulse 800 nm, 1kHz, 1mJ/pulse Experiment set-up: Automatic 2DIR spectrometer signal beam dump optical chopper k1k1 k pump-probe = k 1 +k 1 +k 2  sample k 1 +k 1 k2k2 monochromator MCT array - - + - - +

6. Result: 2DIR spectrum of fully folded hIAPP 1620 cm -1 ( Beta-sheet) 1645 cm -1 (random coil) 1670 cm -1 ( Beta-sheet)

7. Result: FTIR kinetics Starting from random coil

8. Result: FTIR kinetics 1645 cm -1 (random coil) 1620 cm -1 (beta-sheet) t = 0 min t = 240 min Beta sheet structure grows in and random coil decreases. FTIR intensity change over time

9. Result: 2DIR snapshots Intensities Populations Line shape Vibration Dynamics t=10 mint=150 min t=300 min

10. Result: 2DIR kinetics random coil (1645 cm -1 ) beta sheet (1670 cm -1 ) beta sheet (1620 cm -1 ) Trend of random coil diagonal peak elipticity Elipticity=diagonal width/anti-diagonal width 2DIR intensity change over time

11. Result: comparison FTIR: intensity of beta-sheet peak 2DIR: intensity of beta-sheet diagonal peak (1620 cm -1 ) FTIR 2DIR FTIR 2DIR beta-sheet random coil FTIR: intensity of random coil peak 2DIR: intensity of random coil diagonal peak (1645 cm -1 )

12. Summary: Tentative model of hIAPP folding in D 2 O protofibril newly formed fibril mature fibril hIAPP in random coil T1T1 T2T2 T3T3

13. Faculty Martin Zanni Eric Fulmer Amber Krummel Prabuddha Mukherjee Sang-Hee Shim Feng (Terry) Ding Dave Strasfeld Ann Woys Wei Xiong Acknowledgement

14. How does the Ge pulse shaper work?