The intracellular accumulation of polypeptide chains from cloned genes as non native aggregates - inclusion bodies- has emerged as a problem in biotechnology. One of the few systems for which intracellular intermediates in polypeptide chain folding and aggregation have been identified is the thermostable trimeric tailspike of bacteriophage P22. Mutations which interfere with intermediates in the folding pathway, rather than the native protein, map at over 30 sites in the central region of the polypeptide chain. Use of these mutants has revealed that the inclusion bodies form from an early single chain intermediate in the intracellular folding pathway. Second site suppressors have been isolated which alleviate the tsf folding defects. Suppressors at two nearby sites within the gene appear to act by inhibiting the aggregation pathway. They do not affect the biological activity of the protein once correctly folded. This suggests that the role of some local sequences in the chain is to block off-pathway reactions rather than to stabilize the native protein. Such amino acid sequence information could be of practical significance in the recovery of cloned proteins and of theoretical significance in deciphering the rules through which amino acid sequences encode protein conformation. This proposal focuses on elucidating the mechanism of action of the second site suppressors by preparing urea denatured forms of the purified tsf, sutsf, tsf / sutsf and wild type proteins and comparing their refolding in vitro. These experiments will define whether the suppressors act through intrachain interactions, or require interactions with cellular components such as chaperonins.
