טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentAnati Rina
SubjectProgress in Determination of 3D structures of Proteins
Involved in Manganese Functions in Photosynthetic
Organisms
DepartmentDepartment of Chemistry
Supervisor Professor Noam Adir


Abstract

 We are interested in the roles and interactions of two proteins involved in manganese functions in photosynthetic organisms and we describe here our progress in the crystallization and determination of the three dimensional structures of these proteins: Manganese Stabilizing Protein and Manganese Transport Protein

 The MSP of PS II was purified from spinach photosynthetic membranes. The MSP was crystallized in the presence of calcium. Despite the apparent purity of the isolated protein, the crystals grew to only about 0.05mm in their largest dimension. The MSP was analyzed to identify possible sources of protein heterogeneity that could hinder crystal growth. Tandem reverse-phase HPLC/ electronspray ionization mass spectrometry analysis of the MSP revealed a molecular mass of 26535, as expected for mature MSP, indicating the absence of heterogeneities due to covalent modifications. MALDI mass spectroscopy was utilized to identify heterogeneities in the MSP oligomeric state. These measurements showed that purified MSP in solution is a mixture of monomers and dimers, while solubilized MSP crystals contained only dimers. Size-exclusion chromatography and dynamic light scattering were used to probe the effect of the crystallization conditions on the MSP. Based on the results reported in this study, a model is presented which details the effect of oligomeric heterogeneity on the inhibition of MSP crystal growth.

 In Synechocystis sp. PCC 6803, uptake of manganese is carried out by two high affinity manganese transport systems. Three structural genes for a manganese ABC (ATP Binding cassette)-type transporter system have been previously identified, the first such protein complex for manganese identified in any organism. Of the three protein components encoded by these genes, MntC encodes the periplasmic solute binding protein (SBP). The mntC gene from Synechocystis 6803 was cloned into the vector pET-3XC for expression as a soluble protein without the lipid anchoring domain. The MntC protein was isolated, purified and crystallized.  The crystals diffracted to a maximum resolution of 2.6 Å. Analysis of the diffraction pattern using either DENZO or Mosfilm indicated that the MntC crystals belong to a hexagonal space group with unit cell dimensions of 126Å x 126Å x 88Å. A number of native data sets, 100% complete to 3.0Å were collected.

Phasing by molecular replacement using the Streptococcus pneumoniae PsaA structure, a protein with about 30% homology, is being attempted.