College Honors Program

The Influence of Surface Residues on the Structure and Activity of a Salt-Dependent Halophilic Enzyme

Date of Creation

5-31-2023

Document Type

Campus Access Only

Department

Chemistry

First Advisor

Kenneth Mills

Abstract

Halobacterium salinarum is an archaeal microorganism that grows at high salt concentrations. The DNA PolII protein in Halobacterium salinarum contains an intein that interrupts the sequence of the mature 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 Halobacterium salinarum intein requires high salt to function with at least 1.5 M NaCl. By comparing this intein to those from other non-halophilic extremophiles, we identified nine surface residues that were negatively charged in the Halobacterium salinarum intein but either positive or neutral in the homologs. We found that a mutant intein with these nine changes can splice at 0.5 M NaCl. We were further able to narrow this activity to two specific residues, suggesting that only two amino acid changes can trigger a substantive folding and activity difference. In addition, we used native tryptophan fluorescence to study the folding of the wild-type and mutant inteins as a function of salt concentration; the mutant is well folded at relatively low salt. We would like to thank the National Institutes of Health, NIGMS (Grant 1R15GM132817-01) for supporting our research.

Comments

Reader: Christine Hagan

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