Characterization of Novel HydX Protein and its Involvement in the Formation of the Active Site in [Fe-Fe] Hydrogenases Open Access

Klass, Sarah Harrington (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/b2773v766?locale=en
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

About this Honors Thesis

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files