Assessment of Protein Conformation via Diazirine-Promoted Oxidation

Protein structure elucidation is essential for proteomics as it sheds insight into protein function. Photo-affinity label-ing (PAL) utilizes photoreactive molecules that derivatize solvent-accessible sites in the target protein and the prod-ucts of this reaction can be used to deduce conformational information. In this work, we devised a novel oxidation process instigated by UV irradiation of aromatic diazirines. We initially explored the labeling of the peptides ACTH (1-14) and melittin. In both cases, a highly selective oxidation occurred only on methionine (Met) and tryptophan (Trp). PAL on ubiquitin, followed by top-down MS sequencing, also confirmed the oxidation on the single methio-nine. The optimum condition for the diazirine-promoted oxidation (DPO) was using 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid (TDBA) as a diazirine precursor with short UV irradiation of 2 minutes. To assess whether this labeling could be utilized to evaluate protein conformation, we conducted differential PAL studies between na-tive and urea-denatured forms of ubiquitin and cytochrome c, as well as holo- and apo-myoglobin. Oxidative labeling could not distinguish between native and denatured states of ubiquitin, likely due to only subtle changes in solvent accessibility of the methionine residue. In contrast, this technique successfully distinguished each conformation of cytochrome c and myoglobin with p < 0.05 and p < 0.005, respectively. Bottom-up MS analyses also provided residue-level information. The oxidative labeling also successfully probed the burial of Trp60 in the early stage of amyloid forming. This novel oxidative labeling is highly promising when combined with well-known carbene labeling. The resulting dual mechanism labeling workflow could be an ideal candidate for analyzing protein-protein interactions, which are crucial for therapeutic interventions.