| Research Abstract |
On the biological membranes, many different protein complexes are functioning and they have strong relations each other. Recent progress of the genome project and the development of the related molecular biology technique revealed that the interaction among these protein complexes are very intimate not only on the function but also on the biogenesis of them. However, it is not understood very well which kind of interaction control the total function of the multiple complexes and the assembly of the machinery. Here, we tried to clarify the network of the multiple protein complexes on the biological membranes. For this research we focused on the photosynthetic apparatus on the thylakoid membrane. In 1996, the whole genome sequences of the cyanobacteria, the potential origin of the chloroplast of higher plants, were revealed by the researchers of Kazusa DNA research laboratory Japan and the information of the proteins at the amino acid level for the whole machinery of the photosynthetic c
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omplexes of this bacteria has become available. We here adopted this advantage for our study.
Additionally, we chose FィイD20ィエD2FィイD21ィエD2 ATP synthase, one of the most complex apparatus on the thylakoid membrane as a research target for the study of the assembly of the molecular machinery.
1. The evaluation of the role of GroEL/ES system of cyanobacteria for the protein complex assembly and the protein expression in E. coli
GroEL/ES system facilitates folding of non-native proteins in vivo and in vitro. The most well-studied GroEL/ES complex is the one from Escherichia coli. The crystal structure of this complex is already reported. The complex has a cylindrical cavity which can accept the unfolded peptides. The Similar protein complex was found in Synechocystis PCC6803 cell (Plant Moi. Biol. (1992) 18, 327-336). The larger subunit of the complex (HSP64) had the homology with GroEL and was assigned as GroEL- related chaperonin. As there are some reports that the bacterial GroEL/ES system could assist the well expression of the introduced proteins, we here examined whether the GroEL/ES complex from Synechocystis can assist the expression of the certain protein of cyanobacteria in E. coli cell.
For this purpose, the genes for GroEL, GroEL2 (GroEL homolog), and GroES were obtained from total DNA of Synechocystis PCC6803 by PCR method and the expression vectors for each of them were constructed. As we already had several proteins from Synechocystis cell, of which expression in E. coli was very difficult, we here tried the co-expression of these proteins with the GroEL/ES system of cyanobacteria. Interestingly, although most of these proteins were expressed as inclusion body in E. coil cell, the expression of the one of these proteins from cyanobacteria was strongly affected by the co-expression of cyanobacterial GroEL/ES system, thus giving the soluble protein.
2. On the assembly of the ATP synthase
To understand the functional biogenesis of the chloroplast ATP synhtase, we here studied the interaction between γ subunit or ε subunit and αィイD23ィエD2βィイD23ィエD2 core complex. For this purpose, a couple of mutant γ subunit were expressed as recombinant proteins. To asses the effects of these mutations, the ATPase active chimeric complex was successfully reconstituted with the recombinant γ subunit and the α and β subunits from thermophilic bacteria PS3. By using this system, we could assign several important region for the regulation of the enzyme activity. In case of ε subunit, the authentic αィイD23ィエD2βィイD23ィエD2γcomplex prepared from chloroplast coupling factor ATPase were used for the study of the inhibition effect. We could find several important charged residues on the putative α-helix structure (predicted from the homology with bacterial ε subunit) for the function.
Furthermore, we investigated the regulatory mechanism of chloroplast ATP synthase in light of the interaction between the enzyme and the regulator protein, thioredoxin. From the study, we found the novel interaction between them which will raise the conformational change of the target region. This thioredoxin-induced conformational change must be important for the efficient activation of the enzyme. Less
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