CRYSTALLIZATION AND PRELIMINARY X-RAY STUDIES OF PLASTOCYANIN FROM SILENE EXPRESSED IN E. COLI. T. Inoue*, M. Gotowda*, K. Hamada*, T. Takabe** & Y. Kai*.*Department of Applied Chemistry, Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan, **Department of Chemistry, Faculty of Science and Technology, Meijo University, Tenpaku-ku, Nogoya 468, Japan.
Plastocyanins are type I copper proteins with a single polypeptide chain (10.5KDa). They have been found in a variety of higher plants and algae where they function in photosynthetic electron transport (Sykes, A. G., et al., 1993). Plastocyanins are unique proteins among the blue copper proteins showing the two different electron transfer (ET) sites (Sykes, A. G., 1991, Qin, L. & Kostic, N. M., 1993). However, it still remains necessary to understand further the precise specificities of the two sites, the nature of the binding, and the intramolecular ET to and from in particular the remote site with different redox partners. In order to make clear the reletionships between their functions and structures, we have carried out the study on Crystallization and Preliminary X-ray Studies of Plastocyanin from Silene expressed in E. Coli (PCSIL). PCSIL has been crystallized in a form suitable for X-ray diffraction analysis by macroseeding method using ammonium sulfate as a precipitant in acetate buffer (PH=5.5). These crystals belong to space group P3221 with lattice parameters a=b=76.6 Å and c=65.5 Å, indicating an asymmetric unit containing two plastocyanin molecules. The crystals diffract up to at least 2.0 Å resolution. 44196 diffraction data were observed, from which 11515 were unique, in the resolution range 15.0-2.0 Å, with an Rmerge of 6.0%. Molecular replacement method was applied to solve the crystal structure with AMoRe in CCP4. Rigid-body refinement of the model and subsequent refinement using molecular dynamics were carried out with XPLOR, leading to a current R factor of 17.6%, for the diffraction intensities up to 2.5 Å resolution.
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