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Structural and Functional Characterization of the Sco Protein from Thermus thermophilus
Author(s) -
Lopez Liezelle,
HunsickerWang Laura
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.608.2
Subject(s) - thermus thermophilus , chemistry , tcep , cysteine , crystallography , organic chemistry , catalysis , phosphine , biochemistry , enzyme , escherichia coli , gene
The Sco family of proteins has been implicated in the assembly of cytochrome c oxidase (Complex IV) of the respiratory electron transport chain, but its function is still under debate. Tt Sco has been shown to bind to Cu (I) and Cu (II) ions. However, it is also structurally similar to thioredoxins and has been shown to be able to reduce the disulfide bond present in the Cu A site of cytochrome c oxidase prior to copper insertion. To provide evidence of Sco's role in the assembly of the Cu A‐ center, single cysteine to serine mutants, Tt Sco‐C53S and Tt Cu A ‐C149S, were first produced. Tt Cu A was conjugated to a chromophore that is released upon reaction with Tt Sco. The two proteins were reacted and monitored via UV‐Visible spectroscopy in a 1:1 ratio at 412 nm to monitor formation of a mixed disulfide intermediate (MDI) between the two. Tt Sco and Tt Cu A have been reacted to form the MDI at different stoichiometric ratios and to probe the kinetics of formation of the MDI. Our results indicate much higher production of MDI with Sco in excess. Our second aim is to crystallize the mixed disulfide intermediate to visualize and better understand the interactions between Tt Sco and Tt Cu A . Cation exchange chromatography has shown the most success in separating MDI from unreacted starting materials. A successful condition for crystallization of the MDI has been found and consists of ammonium sulfate and polyethylene glycol 8,000. Lastly, the effects of copper binding on the relative stability of apo and holo forms of Tt Sco have been studied via chemical denaturation. Interestingly, preliminary data suggests that holo‐ Tt Sco is slightly less stable than apo‐ Tt Sco. Further studies aim to investigate the effect of the copper‐ligating His139 on stability of the protein, both with and without metal bound to gain more insight into Tt Sco's ability to bind metals and the role metal binding plays in its function. Support or Funding Information Welch Foundation