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 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, ArcLoop Soto, C.. Nat Rev Neurosci 4, (2003). Glutamine is Important in all of these Diseases! PolyQ Structures from the Zanni Group

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

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 Other Results: Z-Q-OH and Z-Q-NHMe Assigned Conformations C Å C Å C5/C8 0.1 kJ/mol Backbone → Side-Chain C5, 2.16 Å C Å C5/C kJ/mol Backbone → Side-Chain C Å C Å C9/C kJ/mol Side-Chain → Backbone C Å Amide-Stacked/C kJ/mol “Cap Influenced”

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

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

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

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

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

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

12 Major Findings: Ac-Ala-Gln-NHBn forms type I β-turn S. Xiang, et. al., Biochem. 36 (1997), 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 P.N. Lewis, et. al., Biochim Biophys Acta, 303 (1973), (-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

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 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

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

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

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

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

Z-Glutamine-OH Conformer A: C5/C8 C Å C 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”

Z-Glutamine-OH Conformer B: C9/C5 C Å C 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”

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” C Å 21 Hydride Stretch Amide I & II OH Bend † “Bleed Through”

22 Ac-Gln-NHBn, Conformer A: C5/C8 Extended Backbone C Å C Å C5/C kJ/mol Extended Backbone Sidechain-Backbone

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

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

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 C8 Hydrogen Bonds: -OH vs. –NHMe Z-Gln-NHMe: Conformer AZ-Gln-OH: Conformer A C Å C Å C Å C Å 3350 cm -1 Δ ~ 150 cm cm -1 Δ ~ 450 cm -1

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 Full Crystal Structure Overlap

29 X

30 X

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32 X

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35 X

36 X

37 X

38 X

39 X

40 X

41 X