Characterization of Novel HydX Protein and its Involvement in the Formation of the Active Site in [Fe-Fe] Hydrogenases Public
Klass, Sarah Harrington (2013)
Published
Abstract
The novel HydX protein is expressed during anaerobic respiration in organisms containing [Fe-Fe] hydrogenase enzymes. In anaerobic conditions, protons are used as a final electron acceptor and are reduced at the active site of hydrogenases to produce H2 (g). Although no prior research has been done on HydX, the structure and function of three accessory proteins (HydE, HydF, and HydG) known to be involved in the maturation of the active site in [Fe-Fe] hydrogenases have been identified, however, the complete maturation mechanism has yet to be elucidated. The gene sequences for the known accessory proteins directly flank the hydx sequence on either side and are also expressed during anaerobic along with the hydrogenase enzyme HydA. In this thesis the structure and function of HydX was explored to determine if it too had a role in the formation of the active site by acting as a scaffold or transport protein. First, a non-redundant BLAST search of the hydx sequence was performed, surprisingly, no homologs in structure or sequence were found. The CD spectrum of HydX indicated a primarily alpha-helical structure with a Tm of 48.5ºC. Size exclusion chromatography of the HydX protein indicated a trimeric oligomeric state. NMR spectroscopy revealed an apparently folded tertiary structure with some flexible regions. Pull down experiments with three known accessory proteins indicated possible protein-protein interactions between HydX and HydG.
Table of Contents
Table of Contents
…………………...………………………………………………………………………….Page #
List of Figures………………………………………………………………………………....1
Abbreviations Used…………………………………………………………………….…….2
Background……………………………………………………………………………....3 - 10
Specific Aims………………………………………………..….…..……………….……….11
Experimental Procedure……………………………………………….………….12 - 20
Results and Discussion………………………………………………………...…21 - 31
Conclusion……………………………………………………………………………………….32
Appendix………………………………………………………….………………………33 - 35
Literature Cited….…………………………………………………..………………36 - 40
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