Assessment of Protein Conformation via Diazirine-Promoted Oxidation of Methionine and Tryptophan Residues

Photoaffinity labeling (PAL) utilizes photoreactive molecules to derivatize proteins in a solvent-accessible dependent manner. The site and frequency of the products of this reaction can be used to garner insights into the conformation of the protein. In this work, we document and characterize a novel oxidation process instigated by UV irradiation of aromatic diazirines. Diazirine is an increasingly utilized reagent for probing protein conformations, and this product has yet to be documented. We initially assess the selectivity of the chemical reaction and find that it is highly selective to methionine (Met) and tryptophan (Trp) residues. We next examine whether this oxidative labeling can be utilized to evaluate protein conformation. We assess native and urea-denatured ubiquitin and cytochrome c, holo- and apo-myoglobin, and native and copper-bound β-2-microglobulin. In all surveyed protein systems but ubiquitin, this diazirine-promoted oxidation readily differentiates between the two protein conformations.