1. 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

2. 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

3. 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.

4. 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

5. 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)

6. Instrumentation

7. 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

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

9. 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)

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

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

12. 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)

13. 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+