OSU Conference 2010: Symposium on Metal Containing Molecules An Unexpected Binding Motif for Metal Di-Cation Complexation with Peptides IRMPD Structure Determination Robert C. Dunbar Case Western Reserve University Jeffrey D. Steill FOM Institute for Plasma Physics Nicolas Polfer University of Florida Giel Berden Jos Oomens and University of Amsterdam

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

Working with the Complexes 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.

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

A Zoology of Metal-Ion Dipeptide Complexes Ag+ Na+ Charge Solvated OOR Charge Solvated NOR Structure motifs all identified by IRMPD and supported by calculated thermochemistry. Metal-ion binding to Carboxy oxygens Amino nitrogen Phenyl ring - + Ba2+ Ca2+ Charge Solvated, Caged OORR Zwitterion ZOOR

Look at Stronger Binding Metals Charge Solvated OORR Move up in metal ion binding strength: Dramatic change in spectral pattern between calcium and magnesium

Iminol Tautomerism of Amide The amide linkage can tautomerize to the iminol form (like keto/enol tautomerism) but this is unusual in condensed phase. Metal ion binding to the amide nitrogen normally observed in solution only accompanied by deprotonation (high pH) Spectroscopic signatures: No amide II (1550 cm-1) OH bend (1430 cm-1)

Rearrangement to the Iminol Tautomer Amide II OH bend DFT Charge Solvated DFT Iminol Tautomer

What about PheAla? PheAla results quite similar to PhePhe CS CS PheAla results quite similar to PhePhe Except the Mg2+ spectrum has some presence of CS CS CS Im CS CS Im

Why is Iminol Binding Interesting? Notable points about iminol tautomer binding for Mg2+, Ni2+ Metal—NAmide bond well known (usually Ni2+ or Cu2+) for deprotonated amide linkages in peptides, but novel as the iminol tautomer with proton retained Metal-amide-nitrogen bond novel for Mg2+ (prefers oxygen ) Ex Examples of deprotonated amide binding Hattori, Tanaka, Fukai, Ishitani and Nureki, Nature 448, 30 (2007) Amide Mg2+ oxygen binding site: Magnesium transporter protein Oxytocin: Wyttenbach, Liu and Bowers, JACS 130, 5993 (2008) Guerrieri et al., J. Biol. Inorg. Chem. 14, 361 (2009)

Conclusions The iminol binding mode is confirmed and dominant for Ni2+ and Mg2+ (also Co2+) with PhePhe and PheAla Iminol binding with a metal-nitrogen bond is not too surprising for Ni2+, but unprecedented for Mg2+ Might encourage condensed-phase chemists to look for amide-nitrogen binding of Mg2+