A Protein Folding Nucleus in the Gas Phase Jessica Thomas and David Pratt University of Pittsburgh Michel Mons, François Piuzzi, Eric Gloaguen, and Benjamin Tardivel Laboratoire Francis Perrin

Motivation  Poupon and Mornon 1 : Folding Nuclei: segments of proteins that fold first  Molinari and Coworkers 2 : Identified folding nuclei in β-lactoglobulin  What intermolecular forces are found in a folding nucleus? (And what does this tell us about protein folding?) Samples = segments of WYSLA from β-lactoglobulin  Acetyl- and –NH 2 caps simulate longer chains 1 A. Poupon and J.-P. Mornon. FEBS Letters (1999) 452, L. Ragona, et al. Biochemistry (2002) 41, 2786.

Experimental  Sample Preparation: Laser ablation  Spectroscopic Methods: UV ionization with mass spec UV/IR hole burning S0S0 S1S1 IR UV

Laser Ablation  Reduces decomposition of large biomolecules  Laser: 2 nd harmonic of Nd:YAG  Graphite matrix

UV/IR Lab Mini YAG UV laser Ablation OPOYAG Laser IR laser Prism creates second UV path through chamber Excimer LaserDye Laser Chambers: Jet and MS Detection

 Acetyl-Tryptophan(W)-Tyrosine(Y)-Amide Two UV chromophores Results: WYp

WYp Ionization  Trp gives broad UV absorption spectrum with sharp features  Use UV spectrum to identify electronic transitions for UV/IR hole- burning Wavenumbers (cm -1 ) Intensity (arbitrary units)

C7 C10 C5 WYp UV/IR  Five N-H bonds, one O-H bond  Possible intramolecular interactions Within backbone: C5, C7, C10 Terminus: NH 2 sym and antisym Involving rings: N-H··· π, O -H··· π Other: free N-H, free O-H  Interactions cause shifts in IR spectrum

C7 or C10 IR Spectra of WYp C5 or N-H··· π (one band) C5 and/or N-H··· π (Two bands) N-H of indole or antisym C10 Bound O-H C7 Conformer A Conformers A + B N-H Stretch RegionO-H Stretch Region Preliminary assignments based on previous results: Chin, et al. JACS (2005) 127, 707. Chin, et al. JACS (2005) 127,

C7 Initial Assignment of WYp C10 Conformer A Conformer B (2) C7C10 N-H··· π (2) N-H··· π Free N-H of indole Bound O-H Conformer A Conformer B

Experimental & Calculated Spectra Conformer A Conformers A & B Calculated 2 Double C7 M05-2X/6-31g Scaled by Calculated 1 Straight Chain M05-2X/6-31g (d,p) Scaled by Calculations in progress

Results: WYSp  Acetyl-Tryptophan(W)-Tyrosine(Y)-Serine(S)- Amide Same two UV chromophores

WYSp UV/IR Experimental Red shifting due to H-bonds Calculated 1 M05-2X/6-31g Scaled by NH 2 symNH 2 antisym O-H of Tyr O-H of Ser N-H of indole (3) Backbone N-H Calculations in progress

Conclusions  Presence of serine changes backbone configuration  Larger molecules tend to form fewer conformers Tryptamine = 7, WYp = 2, WYSp = 1  Peptide acts as a folding nucleus due to H-bonds

Ongoing and Future Work  Adapt ablation for Pittsburgh labs vibrationally and rotationally resolved spectroscopy  WYSLp Identified parent molecule via mass spec Signal did not last long & high ablation power needed Significant fragmentation  Related Molecules [[[Tyr-Ser]-Leu]-Ala] (narrower spectrum without Trp?)

Acknowledgements  Sophie Herbeaux (undergraduate, Université Paris-Sud) for assistance in the lab.  CEA (France), University of Pittsburgh, and NSF for financial support of the project and my visit to the Laboratoire Francis Perrin.

Tentative Assignment of WYSp N-H of indoleBound N-H backbone 3Free N-H backbone 2N-H··· π w/ indole N-H backbone 1Free O-H of SerDoubly H- bonded NH 2 antisym- O-H of TyrH-bonded

Ablated WYSp: Mass Spec WYSp WYSp – 30 amu Oil in chamber Smaller fragments

Ionization/Fluorescence Lab Chambers: Jet and MS Dye LaserYAG Laser PMT, camera & Monochrometer Excimer LaserDye Laser Mini YAG Electronics Computers & Oscilloscope Ablation UV laser, Chambers, and Detection Timing Display and Storage UV/UV Hole Burning 3 rd harmonic pumps coumarin 540A General Valve nozzle, 300 μm ~10 -6 mbar 3 – 6 bar Ar