Abstract 1268 The influence of surface residues on the structure and activity of a salt-dependent halophile enzyme

Halobacterium salinarum (H. salinarum) is a salt-loving archaea that grows at high salt concentrations. H. salinarum contains an intein that interrupts the DNA Pol II protein. Inteins mediate the process of protein splicing, a post-translational modification in which the intein is self-catalytically removed from two flanking extein sequences and the exteins are ligated together. The in vitro protein splicing activity of the intein requires at least 1.5M NaCl to function. To identify the cause of salt-dependent splicing, we compared this intein to those from other non-halophilic extremophiles and identified nine surface residues that were negatively charged in H. salinarum but positive or neutral in the other inteins. We found that a mutant intein with nine substitutions can splice at 0.5 M NaCl. We were further able to narrow this improved activity to two specific residues, suggesting that only two amino acid changes can trigger a substantive folding and activity difference in the protein. In addition, we used native Trp fluorescence to study the folding of the inteins as a function of salt concentration to determine if the folding of the protein is correlated with the splicing activity. We found that the mutant is well folded at relatively low salt, whereas the wild type is only folded at higher salt concentrations. We would like to thank the National Institutes of Health, NIGMS (Grant 1R15GM132817-01) for supporting our research.