Potential artifacts in using a glutathione S-transferase fusion protein system and spin labeling electron...

Site-directed spin labeling electron paramagnetic resonance methods have been an important tool in studying protein-protein interactions. Labels are often attached to a cysteine residue, and spectra are acquired with and without binding partner(s) to provide information on the binding. This requires a knowledge of the label location which is simplified if the label remains faithfully attached to the designated residue in the complex. We report a system where this is not the case because the label was extracted by dialysis-resistant glutathione molecules. Once this artifact is identified, spectral subtraction provides a solution for meaningful data interpretation.