1. 1 The Inherent Conformational Preferences of Glutamine-Containing Peptides: The Role for Side-Chain Backbone Hydrogen Bonds Patrick S. Walsh 1, Jacob C. Dean 1,2, Carl McBurney 3, Samuel H. Gellman 3, Hyuk Kang 4 and Timothy S. Zwier 1 1 Deparment of Chemistry, Purdue University 2 Department of Chemistry, Princeton University 3 Department of Chemistry, University of Wisconsin- Madison 4 Department of Chemistry, Ajou University TD08

2. 2 Biological Motivation: Why Glutamine? Amyloid Fibrils (β-sheet): Different Proteins and different Regions of the Brain Alzheimer’s Disease Huntington’s Disease and Polyglutamine diseases Parkinson’s Disease Huntington’s Disease: htt protein 40 Q: Diseased PolyQ Structures: β-sheet (Anti-Parallel) β-hairpin β-arc Sidechain dominant HB (Solvent, Sidechain, or Backbone) Buchanan, L.E., et al., PNAS, 111, 16, 2014. ArcLoop Soto, C.. Nat Rev Neurosci 4, 49-60 (2003). Glutamine is Important in all of these Diseases! PolyQ Structures from the Zanni Group

3. 3 Physical Chemist’s Motivation: What is the Sidechain doing? What are the inherent structural preferences of glutamine residues? Types of Hydrogen Bonds? Sidechain-to-Backbone? Backbone-to-Backbone? Side-Chain → Backbone vs. Backbone → Backbone C5 C7 C9 C8 C5 C10 C8 C10

4. Experimental Methods: Conformation-Specific Spectroscopy RIDIR Resonant Ion-Dip Infrared Spectroscopy M (S 0 ) M* (S 1 ) M + + e - M (S 0 ) M* (S 1 ) M + + e - R2PI Resonant Two-Photon Ionization 20 Hz 10 Hz Δt=200ns 20 Hz Sample Prepared via Laser Desorption Calculations: M052x/6-31+g(d) 4 Turbo MCP Turbo TOF Tube

5. 5 Other Results: Z-Q-OH and Z-Q-NHMe Assigned Conformations C5 2.12 Å C8 1.97 Å C5/C8 0.1 kJ/mol Backbone → Side-Chain C5, 2.16 Å C8 1.73 Å C5/C8 1.85 kJ/mol Backbone → Side-Chain C5 2.31 Å C9 1.97 Å C9/C5 5.38 kJ/mol Side-Chain → Backbone C5 2.39 Å Amide-Stacked/C5 0.00 kJ/mol “Cap Influenced”

6. 6 Ac-Gln-NHBn: UV Spectrum Ac-Gln-NHBn 3 Main Conformers!!! P.S. Walsh, et al., Manuscript in preparation, (2015). ?

7. 7 Assigned Conformations: Ac-Gln-NHBn C8 1.96 Å C5 2.11 Å C5/C8 1.54 kJ/mol Extended Backbone Sidechain-Backbone Conformer A C8 C7 C8 2.24 Å C7 1.92 Å C7 1.99 Å C7/C7/C8/π 0.57 kJ/mol Turned Backbone Sidechain-Backbone Backbone-Backbone C7 Conformer C Maximum Number of Hydrogen Bonds

8. 8 Ac-Gln-NHBn, Conformer B: C7/π Turned Backbone C7 1.98 Å C7 1.98 Å π π C7/π 0.00 kJ/mol Turned Backbone Sidechain-Backbone

9. 9 Expanding the Backbone: UV Spectra of Ac-Ala-Gln-NHBn Ac-Ala-Gln-NHBn 1 Conformer!!!

10. 10 Ac-Ala-Gln-NHBn: C10/C7/π Turned Backbone π C7 1.97 Å C10/C7/π 0.00 kJ/mol Turned Backbone Backbone-Backbone Sidechain-Backbone C10 2.02 Å C10 C7 π

11. 11 Natural Extension from Di- to Triamide: Starting a β-turn C10 C7 1.97 Å π C7 1.98 Å π Ac-Gln-NHBnAc-Ala-Gln-NHBn Glutamine Hydrogen Bonding and Structure Similar in Both Molecules!!!

12. 12 Major Findings: Ac-Ala-Gln-NHBn forms type I β-turn S. Xiang, et. al., Biochem. 36 (1997), 4768-4774. PDB ID: 1AF2, Cytimdine Deamidase Crystal Structure (Residues 9-12, FAQL) Ac-AQ-NHBn Gas-Phase Structure F(AQ)L- Crystal Structure C10 Type I β-Turn φ i+1 ψ i+1 φ i+2 ψ i+2 -60-30-900 P.N. Lewis, et. al., Biochim Biophys Acta, 303 (1973), 211-229. (-70.0, -13.3) (-73.0, -11.0) (-57.0, -21.7) (-98.4, 1.7) Gas-Phase Structure Accurately Reproduces Natural Type I β-Turn

13. Conclusions and Next Steps 13 Conclusions: Sidechain-to-backbone hydrogen bonding is dominant. Ala-Gln forms a β-turn/type I Important to β-hairpin structures! Next Steps Go Bigger Neutral: Ac-QQ-NHBn Ions: Key Aβ, hIAPP, and PolyQ Fragments Model Development Accurate models for predicting spectra Understand solvent effects! Understand role of sidechain-to- sidechain hydrogen bonding NEW Stabilizing Interactions! 0.00 kJ/mol 2.08 kJ/mol Amide-Stacked! 2.65 kJ/mol Ac-Gln-Gln-NHBn

14. 14 Acknowledgements The Zwier Research Group: Prof. Timothy Zwier Dr. Christian W. Müller Bochum Dr. Jacob C. Dean Princeton Dr. Samuel Gellman UW-Madison Brian Fisher Carl McBurney Dr. Evan G. Buchanan NIST Boulder Labs

15. OH Torsional Potential: Glycine Trans-OH Cis-OH 15

16. Z-Glutamine-NHMe: UV Spectra 16 Z-Glutamine-NHMe

17. Z-Glutamine-NHMe: C5/C8 C5/C8 0.1 kJ/mol Extended Backbone Side-Chain → C-term. Backbone → Side-Chain C5 2.12 Å C8 1.97 Å 17 NH Stretch Amide I & II C=O Stretch NH Bend

18. Z-Glutamine-OH: UV Spectra 18 Z-Glutamine-OH Three conformations found in the expansion. More diversity coming from the –OH? A C B

19. Z-Glutamine-OH Conformer A: C5/C8 C5 2.16 Å C8 1.73 Å C5/C8 trans-OH 1.85 kJ/mol Extended Backbone Side-Chain → C-term. Backbone → Side-Chain 19 Hydride Stretch Amide I & II OH Bend † “Bleed Through”

20. Z-Glutamine-OH Conformer B: C9/C5 C5 2.31 Å C9 1.97 Å C9/C5 cis-OH 5.38 kJ/mol Extended Backbone Side-Chain → N-term. Side-Chain → Backbone 20 Hydride Stretch Amide I & II OH Bend † “Bleed Through”

21. Z-Glutamine-OH Conformer C: Amide-Stacked/C5 Amide-Stacked/C5 cis-OH 0.00 kJ/mol Extended Backbone Side-Chain → N-term. “Cap Influenced” C5 2.39 Å 21 Hydride Stretch Amide I & II OH Bend † “Bleed Through”

22. 22 Ac-Gln-NHBn, Conformer A: C5/C8 Extended Backbone C8 1.96 Å C5 2.11 Å C5/C8 1.54 kJ/mol Extended Backbone Sidechain-Backbone

23. 23 Ac-Gln-NHBn, Conformer B: C7/π Turned Backbone C7 1.98 Å C7 1.98 Å π π C7/π 0.00 kJ/mol Turned Backbone Sidechain-Backbone

24. 24 Ac-Gln-NHBn, Conformer C: C7/C7/C8/π Turned Backbone C8 2.24 Å C7 1.99 Å C7 1.92 Å C8 2.24 Å C7 1.92 Å C7 1.99 Å C7/C7/C8/π 0.57 kJ/mol Turned Backbone Sidechain-Backbone Backbone-Backbone

25. Amide-Stacking: Foldamers vs. Natural Residues Ac-γ 2 -Phe-NHMe a Z-Glutamine-OH (+105, -57, +80, -136) Interior N … C: 2.97 Å Exterior N … C: 3.16 Å (-102, +55, -76, +136) Interior N … C: 2.90 Å Exterior N … C: 3.05 Å a James, et al., J. Am. Chem. Soc., 131, 6574 and 14243, (2009). 25

26. 26 C8 Hydrogen Bonds: -OH vs. –NHMe Z-Gln-NHMe: Conformer AZ-Gln-OH: Conformer A C5 2.12 Å C8 1.97 Å C5 2.16 Å C8 1.73 Å 3350 cm -1 Δ ~ 150 cm -1 3142 cm -1 Δ ~ 450 cm -1

27. Experimental Methods: Conformation-Specific Spectroscopy RIDIR Resonant Ion-Dip Infrared Spectroscopy M (S 0 ) M* (S 1 ) M + + e - M (S 0 ) M* (S 1 ) M + + e - R2PI Resonant Two-Photon Ionization IR-UV HB Infrared-Ultraviolet Holeburning M (S 0 ) M* (S 1 ) M + + e - 20 Hz 10 Hz Δt=200ns 20 Hz 10 Hz Δt=200ns 20 Hz Sample Prepared via Laser Desorption Calculations: M052x/6-31+g(d) 27

28. 28 Full Crystal Structure Overlap

29. 29 X

30. 30 X

31. 31 X

32. 32 X

33. 33 X

34. 34 X

35. 35 X

36. 36 X

37. 37 X

38. 38 X

39. 39 X

40. 40 X

41. 41 X