Return to search

Spectroscopic studies of the human copper chaperone for superoxide dismutase : probing the active cluster with selenocysteine variants

Ph.D. / Biochemistry and Molecular Biology / Selenocysteine-containing mutants of human copper chaperone for superoxide dismutase (hCCS) were constructed using intein-mediated peptide ligation. These mutants were studied with respect to their ability to transfer Cu to E,Zn superoxide dismutase (SOD1) and their Cu-binding and X-ray absorption spectroscopic (XAS) properties. Previous studies have shown that three functionally distinct polypeptide domains are present in CCS: the N-terminal domain 1 (D1, residues 1-85) contains the copper-binding MXCXXC motif, domain 2 (D2, residues 86-234) has sequence homology to residues associated with the native SOD1 dimer interface, and the C-terminal domain 3 (D3, residues 235-274) contains a CXC motif. Recent results suggest the formation of a D3- D3 cluster within a dimeric or tetrameric protein and suggest that this cluster may be an important element of the copper transfer machinery. D3 cysteine-to-selenocysteine mutants of wild-type and D1 mutants of hCCS were constructed to investigate the D3 copper cluster in more detail. These mutants display similar activity to wild-type protein. The structure of the Cu centers of selenocysteine-containing mutants as shown by Cu EXAFS is similar to that of wild-type protein, with clear indications of a Cu cluster. Cu and Se EXAFS of these constructs reveal a unique adamantane-like cluster formed between two molecules of CCS at the D3-D3 interface. These results confirm the existence of a D3-D3 copper cluster in hCCS and suggest that a unique copper cluster may exist in this protein.

Identiferoai:union.ndltd.org:OREGON/oai:content.ohsu.edu:etd/258
Date10 1900
CreatorsBarry, Amanda Nell
PublisherOregon Health & Science University
Source SetsOregon Health and Science Univ. Library
LanguageEnglish
Detected LanguageEnglish
TypeText
FormatNeeds Adobe Acrobat Reader to view., pdf, 3064.513 KB
Rightshttp://www.ohsu.edu/library/etd_rights.shtml

Page generated in 0.002 seconds