Structure-function relationships of ricin A-chain

Abstract

This thesis represents e study of the reletionships between the structure and function of the A-chain of ricin. Ricin is one member of a family of ribosome- inactivating proteins (RIPs) that inactivate protein synthesis by modification of the ribosomal RNA. The mechanism of this catalytic effect is unknown. This project was designed to investigate which amino acid residues were important in the mechanism of catalysis. Using site-directed mutagenesis of specific ricin A- chain residues, eight mutants were created for biochemical analysis. Residues were targeted for autogenesis by analysis of the position of conserved amino acids in the ricin 3-D structure. Glul77 was mutated to Lys, Ala and Asp. and Argl8O was mutated to Gin, Ala and Met. The arginine residue at position 29 was altered to an alanine, and a deletion of residues Serl76 to Argl80 was performed. Mutant recombinant ricin A-chain (rRTA) constructs were prepared for in vitro and in vivo expression in the vectors pGEMl and pDS5/3 respectively. Initially, mutants were translated in a cell-free wheatgerm translation system to assess the size of the mutant polypeptides. All the constructs produced polypeptides of the correct else. The N-glycosidase activity of the mutants was then assessed with two methods of analysis using rabbit reticulocyte ribosomes. It was shown that some of the mutants were devoid of detectable activity and some had reduced activity. Mutant constructs were expressed in Escherichia coli in order to isolate protein for quantitative activity measurements. Crude E. coli extracts containing rRTA and rRTA mutants were tested for activity. It was found that the relative activities of mutant proteins produced in E. coli was similar to that seen previously with the in vitro measurements. Soluble, active protein was recovered for a few of the mutant proteins, although no expression was observed from some constructs. Various purification procedures were assessed end ion-exchange chromatography was determined to be most suitable. Mutant D177 was tested for its N-glycosidase activity towards salt-washed yeast ribosomes and related to the activity of wild-type rRTA. It was shown that the activity of D177 was approximately 60 fold lower than that of wild- type rRTA with the majority of the difference being observed with the kcat of the reaction. It was also shown that the data produced in this study correlated with a recently published putative mechanism of action of ricin A-chain