Abstract

The FNR protein is a transcription factor that allows Escherichia coli to undergo anaerobic cellular respiration. It is known to positively regulate the expression ofseveral genes required for anaerobic respiration as well as negatively regulate genes responsible for aerobic respiration. Consequently, FNR is active under anaerobic conditions and inactive under aerobic conditions. Although the tertiary structure ofFNR is unknown, previous studies have indicated that FNR is inactive in the monomeric state and active in the dimeric state. Thus, it is believed that in anaerobic conditions, FNR undergoes a confonnational change from the monomeric to dimeric state. The mechanism involved in going from the monomeric to dimeric state is not completely understood, but it is thought to be triggered by the acquisition ofa [4Fe-4S]2+ cluster in the N-tenninal region ofFNR. The acquisition ofthe cluster causes a confonnational change to be transmitted through the allosteric domain to the dimerization helix resulting in the active dimeric species. Infonnation regarding the environment of amino acid residues in the dimerization helix in both the active and inactive fonns of FNR could be helpful in eliciting a better understanding ofthe dimerization mechanism. Such environmental conditions can be detennined by the fluorescent properties ofthe amino acid, tryptophan. Surface exposed tryptophan residues are expected to have a longer Amax than those buried in the hydrophobic core. In order to gain insight into the environment ofthe amino acids on the dimerization helix we have created tryptophan mutants (LWI46, MW147, MW157, KW163, and KW164) that either lay on or near the helix. The mutants KW163 and KW164 all lie on the periphery ofthe helix while LW146, MW147 and MW157 lie on the helix. Ofthe five mutants, KW163 retained anaerobic activity most similar to that of the wild type indicating that its structure is similar to the wild type protein with the exception ofthe single amino acid substitution. By comparing the fluorescence ofthe active and inactive forms of KW163, we hope to gain a better understanding ofthe dimerization mechanism ofFNR.

Disciplines

Biochemistry

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Biochemistry Commons

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