Characterizing Peptide β-Hairpin Loops via Cold Ion Spectroscopy of Model Compounds John T. Lawler, Andrew DeBlase, Chris Harrilal, Scott A. McLuckey, Timothy S. Zwier

Pancera, M.; et.al. Nature 2014, 514 (7523), 455–461. Why study β-turns? Ubiquitous in biology and often used in drug/synthetic design Turns provide 3D structure via allowing the protein to fold over itself Diener, M. E., et.al., . J. Am. Chem. Soc. 2015, 137 (38), 12369–12377. Pancera, M.; et.al. Nature 2014, 514 (7523), 455–461. Marcelino, A. M. C. , et.al. ,Biopolymers 2008, 89 (5), 380–391.

Steric Effects and Differences in Structure YAPAA DP LP syn anti C11,C17,πNH/C14/trans/FNH/C10/C7OH C5,C11,πNH/C14/trans/FNH/C10/C7OH 3.15 kJ∙mol-1 35.70 kJ∙mol-1 What happens when the residue C-terminal to proline is less bulky? YGPAA

Size and β-turn formation Will increasing the size of the peptide lock in the β-turn ? Will we see the beginnings of beta sheet formation?

Instrumentation Spectroscopy axis MS axis q3 q2 q1 Nano ESI Det 1 qtrans2 qtrans1 Cold Trap Turning Quad 1 Turning Quad 2 Nano ESI

Question 1 Does removing steric hindrance C-terminal to Proline change the proclivity for β-turns?

UV Photofragmentation Spectroscopy YADPGA Reduced number of conformers! YALPGA Changed this from side by side spectra, not really liking it

YADPGA IR-UV Holeburning Spectrum Holeburn Spectrum *IR fixed on 3392.4 cm-1 YADPGA IR-UV holeburning can be used to obtain conformation-specific UV spectra of ions. We fix the IR laser on an infrared transition in the IR spectrum and scan the UV probe. Any UV transitions sharing the same ground state as the IR transition will show a depletion in photofragment signal. Wavenumber (cm-1) One major conformer is seen, minor conformer(s) are likely present

Glycine Position Effecting Conformational Preferences Gly-substituted D-Proline peptides show a reduced amount of conformers (a) YADPGA YGLPAA (a) YALPGA Gly-substituted L-Proline peptides present with an increased number of conformers

IR Gain Spectroscopy of YADPGA C10 Hydrogen Bond *Calculations performed at the B3LYP-GD3BJ/6-31+G* level of theory C10 NH3+ a in addition to the depopulation of the ground state, absorption of a resonant IR photon can lead to intramolecular vibrational energy redistribution (IVR). This redistribution of energy among accessible vibrational levels results in statistical inhomogeneous broadening of the UV spectrum to the red of the electronic origin.  Thus, in IR-UV gain spectroscopy, the UV laser is fixed at a wavelength to the red of the lowest energy electronic origin in the UV spectrum. Vibrational excitation acts to induce UV absorption at this wavelength, resulting in a gain in UV photofragmentation signal. C14 Tyr OH Free NH C14 Hydrogen Bond C10 and C14 H-bonds are diagnostic for β-turn

Question 2 Will increasing the size of the peptide change the proclivity for β-turns?

Enlarging the peptide reduced the number of conformers to one YAADPGAAA UV Spectrum YAADPGAAA YADPGA Enlarging the peptide reduced the number of conformers to one

YAADPGAAA Infrared Spectra Amide II Amide I Hydride Stretch -NH3+ a C101/C102/C7 -NH3+ a C14 -NH3+ a Free NH Acid OH Tyr OH CH Stretches

Calculated Lowest Energy/Best Fit Structures C10 Hydrogen Bond C14 Hydrogen Bond *Calculations performed at the B3LYP-GD3BJ/6-31+G* level of theory

Conclusions Does removing steric hindrance C-terminal to Proline change the proclivity for β-turns? YES, UV action spectroscopy and IR-UV holeburning confirmed a reduced number of conformers when GLY was placed C-terminal to D-Proline In the case of L-Proline the inverse was witnessed, more conformers are present Will increasing the size of the peptide change the proclivity for β-turns? YES, the peptide YAADPGAAA revealed a single conformer that forms the expected beta-turn Will we see the beginnings of beta sheet formation? NO, increasing the size of the peptide did not produce the tell-tale signs of a beta sheet

Future Work Complete Infrared depletion spectroscopy experiments on YAADPGAAA , YADPGA, and YALPGA Do a more exhaustive search of possible structures for each of the experimental peptides

Acknowledgements Jonathan Amy Facility for Chemical Instrumentation National Science Foundation Zwier Group McLuckey Group