Metalloproteins

Activated Water

Carbonic Anhydrase

Activating Water with an Artificial MetalloproteinJacqueline J. Browning, Robert J. Radford, F. Akif Tezcan

Tezcan LabBackground Information Creating the Model Systems

Activity Assays MBPPhen2 Crystals

Metalloproteins are proteins with metal cofactors. We worked with both HPhen1 and MBPPhen2. The phenanthroline (phen) compound on the proteins is a bidentate ligand that binds to metal ions. The metal ions can then be used in the hydrolysis of other molecules.

Protein Purification

MALDI Mass Spectrometry

UV Visible Spectrometry

SDS-PAGE

Testing for Activated Water

The metal ions within a protein framework can induce hydrolysis by creatingactivated water. The high Lewis acidity of the metal ion in a metalloprotein, such ascarbonic anhydrase, lowers the pKa of the coordinated water making OH- morefavored.

Future Work

Carbonic anhydrase is a physiological example of a metalloprotein. It is found inred blood cells and helps produce bicarbonate buffer. The protein has threehistidines that are bound to a zinc molecule which is used to convert CO2 intoHCO3

-.

HPhen1 is the phen labeled G70C mutation of cytochrome cb562. MBPPhen2 is thephen labeled W59C mutation of cytochrome cb562. HPhen1 was chosen due to thestability of the engineered three coordinate metal binding motif. We worked withMBPPhen2 because phen has previously been shown to be located in ahydrophobic pocket. Both three coordinate metals and hydrophobic environmentsare common features in metalloproteins with the ability to activate water and theseproteins allowed us to explore the reaction in a modular fashion.

p-nitrophenyl actate (PNPA) was added tometal bound MBPPhen2 and the formationof p-nitrophenoxide was monitoredsepctroscopically at 400 nm. The slopesremained consistent (~2 x 10-9 ) so themetal ions did not seem to increase the rateof the reaction.

Sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) was performed inorder to test for any impurties in the protein. Thedark color of the protein band is indicative of thehigh concentration of the protein and the lack ofother bands indicates that it has few impurties.

The heme group inboth proteins createsa peak in absorbanceat 415nm that can beused to monitorprotein concentration.We were able todetermine if theprotein was labeledbecause the phengroup created a peakin absorbance at270nm.

Matrix- assisted laserdeprotonization/ionization (MALDI)mass spectrometry ofHPhen1 showed amain peak with anamu of ~12622indicating that theprotein was correctlylabeled and purified.

pKa ~ 9-10 pKa ~ 7- 8

cytochrome cb562

G70C

HPhen1

W59C

MBPPhen2

IPhen

+IPhen +IPhen

Site-directed mutagenasis

The preliminary results indicatethat a combination of both athree cooridnate zinc ion aswell as a properly engineeredenvironment are necessary forwater activation. In the futurewe will engineer a glutamate toact as an acidic residue tofurther lower the pKa of thereaction. Additionally theinteresting structure ofMBPPhen2 (right) mightindicate its possibility forheterogeneous water activation.

Modular and Versatile Hybrid Coordination Motifs on α-Helical Protein SurfacesRobert J. Radford, Phuong C. Nguyen, F. Akif TezcanInorganic Chemistry 2010 49 (15), 7106-7115

Controlled Protein Dimerization through Hybrid Coordination MotifsRobert J. Radford, Phuong C. Nguyen, Treffly B. Ditri, Joshua S. Figueroa, F. Akif TezcanInorganic Chemistry 2010 49 (9), 4362-4369

The Hydrolysis of p-Nitrophenyl Acetate: A Versatile Reaction To Study Enzyme KineticsJ. Anderson, T. Byrne, K. J. Woelfel, J. E. Meany, G. T. Spyridis, Y. PockerJournal of Chemical Education 1994 71 (8), 715

References