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Analysis of exposed cysteines in cortactin by sulfhydryl modification
Author(s) -
Jenness Christopher,
Kruchten Anne E
Publication year - 2009
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.23.1_supplement.883.6
Subject(s) - cortactin , phosphorylation , tyrosine , chemistry , dtnb , mutant , amino acid , biochemistry , tyrosine phosphorylation , biophysics , microbiology and biotechnology , cytoskeleton , biology , cell , enzyme , glutathione , gene
The actin binding protein cortactin is involved in cytoskeleton restructuring and many instances of cancer cell motility. We examined the structure of cortactin and a mutant mimicking tyrosine phosphorylation using 5,5′‐Dithiobis(2‐nitrobenzoic acid) (DTNB). DTNB will form a covalent bond with exposed cysteines, releasing an TNB anion that can be tracked spectrophotometrically at 412 nm. Cysteines, being hydrophobic amino acids, are typically embedded within a protein, which inhibits this binding. We found that wildtype cortactin has approximately two (of three) cysteines exposed, while a cortactin mutant mimicking tyrosine phosphorylation had only one of the cysteines exposed. This difference suggests that cortactin is an intrinsically unstructured protein that oligomerizes or folds when phosphorylated. Further work will mutate individual cortactin cysteines to alanines to determine which are exposed and which regions are folded. This work was supported by a Faculty Start Up Grant to A.E. Kruchten by The Murdock Charitable Trust and by a Linfield College Faculty Student Collaborative Research Award to A.E. Kruchten and C. Jenness.