טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKanteev Margarita
SubjectStructure-Function Investigation of Metal Ion Binding
Proteins
DepartmentDepartment of Chemistry
Supervisor Professor Noam Adir
Full Thesis textFull thesis text - English Version


Abstract

Transition metals such as Zn, Mn or Cu are vital life elements. We are interested in our research to investigate and to determine the 3D structures of metal-ion binding proteins and to investigate the pathways of metal ion uptake and release.

The MntABC transporter from cyanobacteria Synechcocystis 6803 is expressed at sub-micromolar Mn concentrations and. exhibits high affinity and specificity towards manganese ions (Mn). We have previously determined the structure of the MntC solute binding protein (SBP) by X-ray crystallography to 2.9Å (Rukhman et al  2005) and revealed two unique features: a disulfide bond and a significant positive potential around the binding site.

A mutant lacking the disulfide bond exhibited lower affinity towards Mn and increased flexibility.

Arg116 contributes two interesting aspects to the binding. On the one hand the exit to the active site is positive - which may prevent unbinding and loss of Mn from the active site. In addition, this residue forms a polar center with the surrounding residues, thus locking the N and C-terminal domains. This was confirmed by structure determination of the R116A mutant. Based on the biochemical and structural investigations, we proposed a mechanism for Mn binding and release. We suggest that the MntC protein via Glu forms polar contacts with the MntB transmembrane protein. As a result, the C-terminal domain changes conformation, which permits Mn release. The disulfide bond may be reduced by Met237, which permits Mn transport to the cytoplasm. Alternatively, the reduction of the disulfide bond may inactivate the transport by releasing  the Mn to the periplasm where it is used for biogenesis of PSII complex.

Tyrosinase from Bacillus megaterium-TyrBm (EC 1.14.18.1), catalyses the oxidation of monophenol to diphenol and from diphenol to quinone. TyrBm belongs to the family of type 3 copper proteins which coordinate two copper ions by six conserved histidine residues. We determined the crystal structures of monoclinic and orthorhombic TyrBm crystals (Sandovsky et al.2011) and revealed that monoclinic crystals contained only CuA. We analyzed the structures of the three representative members of type 3 copper proteins and found that these proteins have a common motif of adjacent methionine residues. Site directed mutagenesis of the Met revealed that the most active protein M61L did not contain Cu, in contrast to the non active M215L which contained the highest amount of Cu. The crystal structures of the mutants suggest that methionine residues have an important role during catalytic reaction.