SGKamouz Spring 2018

Biochemistry 430

Spring 2018

Junior Biochemistry Major

Research Times

Monday: 2-5 PM

Thursday: 3-5 PM

Note: Times may vary

BIOC 430-01 = 0.25 credit = 4 hours per week.

Proposed Research Project

General Information

Advisor: Laura Moore

Other research student collaborators: Louis Badang, Jenni Eastman

Proposed Project Description (PPD)

Fumarate Nitrate Reductase (FNR) transcription factor is the main anaerobic switch in Escherichia coli bacteria. In the absence of oxygen, the FNR protein exists as a dimer with a [4Fe-4S] cluster and binds to DNA to regulate transcription of genes needed for anaerobic respiration. When oxygen is present, however, the [4Fe-4S] cluster degrades causing FNR to return to its inactive monomeric form. Examination of the crystal structure of Alivibrio fischeri FNR, a close variant to E. coli FNR, suggested that the dimerization and, thus, activation of FNR is dependent upon a salt bridge interaction between the Asp 130 residue and the Arg 140 residue.[1] Site-directed mutagenesis was used to analyze how various mutations at those two particular residues can affect FNR activity. Analysis of double mutants where the 130 and 140 residues were swapped showed that the salt bridge interaction may not be playing a role in FNR dimerization since the double mutants had no FNR activity. Therefore, additional mutations were constructed to study the importance of the 130 and 140 residues in promoting dimerization. Data from β – galactosidase assays carried out on an E. coli reporter strain to measure FNR activity indicate that only Arg 140 is essential for regulating dimerization. Single mutants in which Asp 130 was replaced displayed FNR activity almost the same as that of the wild type FNR while mutations that replaced Arg 140 resulted in almost no FNR activity. Furthermore, alanine and leucine double mutants displayed FNR activity even higher than the wild type FNR activity.

Instruments to be used

Polymerase Chain Reaction Thermocycler

Electroporation Device

References (2 minimum)

1) Volbeda, A., Darnault, C., Renoux, O., Nicolet, Y., & Fontecilla-Camps, J. C. (2015). The crystal structure of the global anaerobic transcriptional regulator FNR explains its extremely fine-tuned monomer-dimer equilibrium. Science Advances, 1(11). doi:10.1126/sciadv.1501086

2) Moore, L. J., & Kiley, P. J. (2001). Characterization of the Dimerization Domain in the FNR Transcription Factor. Journal of Biological Chemistry, 276(49), 45744-45750. doi:10.1074/jbc.m106569200

Research pledge

I, Sobhi Kazmouz, have read the Chem/Bioc 430 course syllabus and understand the general structure and expectations of the research program. The above material was prepared after consultation, and in conjunction with my research advisor.