Post-translational assembly of oligomeric proteins in nature constitutes a non-spontaneous process, and it may require the assistance of mediating binding proteins. This special class of assembly mediators has been denominated """"""""chaperonins"""""""" and it has been detected in chloroplasts, mitochondria, and in a wide variety of prokaryotes including pathogens and human lymphocytes. The emerging evidence suggests that """"""""chaperonins"""""""" are ubiquitous and there is an intense interest in unraveling the precise molecular events by which this class of proteins mediates the assembly of oligomeric proteins in cells. In addressing that problem, during the last granting period, we were able to identify and biochemically characterize to chaperonins (Cpn6O and Cpn10) from the sulfur photosynthetic organism, Chromatium vinosum. Due to structural and functional similarities among chaperonins from C. vinosum and those from E. coli and higher plants, we hypothesize that Cpn6O and Cpn10 are directly involved in the assembly of oligomeric protein complexes, such as RuBisCO, from its individual subunits. The primary objective of the present research proposal is to allow further characterization of the chaperonins from C. vinosum. The primary goal will be achieved through the following strategies: (a) studies on the ability of Cpn 10 homologs to inhibit the ATPase activity of the Cpn6O proteins from C. vinosum and Escherichia coli; (b) study the intracellular location of the Cpn10 homolog in C. vinosum cells by Immunoelectron Microscopy; (c) cloning and expression of the C. vinosum Cpn6O and Cpn10 genes in temperature-sensitive mutant of E. coli which do not have functional chaperonins at the restrictive temperature; (d) nucleotide sequencing and mapping of Cpn6O, Cpn10 and RuBisCO genes from C. vinosum; (e) study the effect of heat shock and other stress conditions on the transcriptional levels of Cpn6O and Cpn10 genes from C. vinosum; (f) development of a cell-free transcription/translational system from C. vinosum to study the assembly of RuBisCO from its subunits. Our broad long-term objective is to provide basic information regarding functional relationships of """"""""chaperonins"""""""" in nature. We anticipate that our proposed studies on Cpn6O and Cpn10 from C. vinosum will shed light on the specific roles of clinically important proteins such as the Mycobacterium leprae 65-kDa-polypeptide which is important during the establishment of leprosy in infected patients, heat shock proteins in general, and a chaperonin (BiP) involved in the assembly of IgG molecules.
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