Abstract
Bacillus subtilis GabR is a transcriptional regulator involved in nitrogen fixation. Previous studies sh.owed that GabR contains a short N-terminal helix-tum-helix (HTH) DNA binding domain and a long C-tenninal aminotransferase domain. The aminotransferase domain has been studied extensively and has been shown to activate the gabTD operon, allowing for the utilization. of y-aminobutyric acid to produce succinate. The HTH domain is believed to repress this pathway when excess nitrogen is present. Even though the domain organization of GabR is known, it is not possible to understand the precise details of how GabR functions as a transcriptional regulator until the crystal structure of GabR is determined. In this study, expression constructs of GabR and t.GabR, a modified version of GabR lacking theN-terminal HTH-domain, were created and small-scale expression tests were perfonned to determine the criteria for optimizing the amount of protein produced. Large-scale expression and purification ofGabR was then performed to obtain reasonably large amounts of purified protein. The future goal of this study is to crystallize and determine the atomic structures of GabR and t.GabR to understand the structural basis of transcriptional regulation by GabR.