Detection of Oxidation and Palmitoylation in Snare Proteins

Abstract

Cysteine residues play an important role for protein localization, function, and structure. Therefore, it is useful to determine the extent of cysteine modification. We have developed a sensitive assay that is able to determine the number of free (reduced) cysteines in proteins. GST-linked proteins are bound to glutathione coated wells. The free cysteines are biotinylated and detected via streptavidin-HRP. We used this assay to study the amount of oxidation and palmitoylation in SNARE proteins (SNAP-25, Syntaxin 1A, and a no cysteine mutant of SNAP-25). Each protein contains 0-4 cysteine residues and results were compared to the background signal obtained from just GST. Reducing agents (Cu2+, Fe2+, Cystine) and oxidizing agents (cysteine and Zn2+), were used to alter the extent of oxidation/reduction of cysteine residues. Alternatively, reduced cysteines were blocked by reactions with NEM or Palmitoyl-CoA. Except for palmitoylation, all reactions could be driven to near completion during a 10 minute incubation on ice. Palmitoylation (without enzymes) required incubation for 1 hour at RT and high doses of Palmitoyl-CoA to palmitoylate >50% of the cysteine residues. This assay is simple, inexpensive, and relative fast, and should allow greater elucidation of the chemistry of cysteine residues in proteins due to its high resolution.