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A method for site‐specific labeling of multiple protein thiols

We present a generic method for the site‐specific and differential labeling of multiple cysteine residues in one protein. Phenyl arsenic oxide has been employed as a protecting group of two closely spaced thiols, allowing first labeling of a single thiol. Subsequently, the protecting group is removed, making available a reactive dithiol site for labeling with a second probe. For proof‐of‐principle, single and triple Cys mutants of the sulphate binding protein of an ABC transporter were constructed. The closely spaced thiols were engineered on the basis of the crystal structure of the protein and placed in different types of secondary structure elements and at different spacing. We show that phenyl arsenic oxide is a good protecting group for thiols spaced 6.3–7.3 Å. Proteins were labeled with two different fluorescent labels and the labeling ratios were determined with UV‐Vis spectroscopy and MALDI‐Tof mass spectrometry. The average labeling efficiency was ∼80% for the single thiol and 65–90% for the dithiol site.