Robert C. Dunbar Case Western Reserve University Nicolas Polfer University of Florida Giel Berden FOM Institute for Plasma Physics Jos Oomens FOM Institute for Plasma Physics and University of Amsterdam OSU Conference 2012 METAL ION BINDING TO POLYPEPTIDES CHARACTERIZED BY IRMPD SPECTROSCOPY METAL-AMIDE NITROGEN BINDING AND THE IMINOL TAUTOMERIZATION

Introduction Metal ions are often bound by peptides  Ion channels  Metal transport and storage  Active sites Gas-phase study of small model peptides can elucidate types of binding sites favored by different metal ions and different side-chain interactions

Metal Ions Bound to Peptide Chains Two basic modes of backbone binding: Amide carbonyl oxygens “Charge-solvated” (CS) Highly coordinated (e.g. octahedral) Alkali metals and Ca 2+, Mg 2+ Amide nitrogens “Deprotonated” or “Iminol” Square planar (may be capped) Ni 2+, Cu 2+

Normal Condensed Phase Behavior Ex Oxytocin: Wyttenbach, Liu and Bowers, JACS 130, 5993 (2008) Prion Protein: Guerrieri et al., J. Biol. Inorg. Chem. 14, 361 (2009) Examples of deprotonated amide binding Cu 2+, Ni 2+ Magnesium transporter protein: Hattori, Tanaka, Fukai, Ishitani and Nureki, Nature 448, 30 (2007) Calmodulin: Chattopadhyaya et al., J. Mol. Biol. 228,1177 (1992) Mg 2+ Ca 2+ Bound oxygens Prion Protein Examples of carbonyl oxygen binding

The Metal-Ion/Peptide complexes are readily introduced into the mass spectrometer by electrospray of metal salt plus peptide from solution. How to probe structures? Computation (DFT spectrum simulation) Infrared spectroscopy Spectroscopy – Can’t do direct absorption spectroscopy, so must resort to some form of action spectroscopy. Photodissociation spectroscopy – plot extent of dissociation vs IR wavelength. Working with the Complexes

Action Spectroscopy and IRMPD InfraRed Multiple Photon Dissociation IR photon typically 0.1 eV Dissociation energy typically 3 eV Many photons delivered by an intense, short laser pulse (IRMPD) M + Trp M + + Trp

Light Source The Free Electron Laser (FELIX) gives  Convenient sweep across the chemically informative IR spectrum  High intensity and energy per pulse  Tight collimation of beam Downside:  Big (very big)  Expensive (very expensive)

Instrumentation

Gas-Phase Dipeptides Charge Solvated Iminol Tautomer DFT OH bend Amide II 2

Two Things About Dipeptide Complexes  Metal ions less active than Ca 2+ show CS binding Metal ions more active than Mg 2+ show iminol binding Break between calcium and magnesium  Magnesium anomaly – Condensed-phase peptide binding of Mg 2+ is CS, but gas-phase dipeptides show iminol binding Try larger peptide ligands?!

Gas-Phase Dipeptides Charge Solvated Iminol Tautomer DFT OH bend Amide II

Larger Peptides

- - Mg 2+ FGG: CS Diastereomers “Diastereomers” -- Almost identical spectra, but OOOP2 is substantially lower in energy

Conclusions:The Magnesium Story  The Magnesium anomaly: magnesium ion commonly binds condensed-phase peptides in the CS (oxygen) mode. But with gas-phase dipeptides we have observed iminol (deprotonated nitrogen) binding.  The present new results with larger peptides FGG and FGGF show CS binding, which resolves the anomaly

Conclusions: Binding to Larger Peptides  Metal ions less active than Mg 2+ show CS binding K +, Ba 2+, Ca 2+  Metal ions more active than Mg 2+ show iminol binding Ni 2+  The Amide II band at cm -1 is a good diagnostic for the existence of CS binding