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Project Progress

Thioredoxins (Trxs) are found in all forms of life, and therefore must have been present in the last universal common ancestor. Trxs serve three types of functions: 1) provision of reducing equivalents to disulfides formed at the active sites of enzymes during turnover; 2) reduction of regulatory disulfide bonds or oxidized cysteine residues in proteins; and 3) participation as a structural component of protein complexes. Trxs provide an excellent system for studying the recruitment of ancient proteins to serve new functions as life became more complex and organisms diversified to occupy different ecological niches. An enormous piece of the puzzle is missing, however, because we know almost nothing about the functions of Trxs in Archaea.

We began this project using the hyperthermophilic Archaeon Archaeoglobus fulgidus. After encountering difficulties in growing the organism, we switched our focus to Halobacterium sp. NRC-1. Halobacterium is an excellent choice for studies of Trx function. It is an extreme halophile. adapted for growth in environments that contain very high salt (optimally 4.5 M NaCl), are exposed to intense sunlight, fluctuate between oxic and anoxic conditions, and are often laden with toxic heavy metals. It is phototrophic and phototactic, using both chemotaxis and buoyant gas vesicles to control its position in brine pools. Halobacterium is easily grown in the laboratory. Genetic manipulations are possible. The complete genome sequence is known. In sum, we can approach the study of Trxs in Halobacterium with all of the tools we can use in E. coli.

The experiments we have planned require purified Trxs, as well as mutant forms of the Trxs that can capture target proteins but are unable to release them. We have cloned genes encoding six Trxs into a Halobacterium expression vector, and are in the process of cloning them into an E. coli expression vector. We are currently optimizing conditions for expression and purification of these proteins.