High-field (275 GHz) spin-label EPR for high-resolution polarity determination in proteins.

The polarity of protein surfaces is one of the factors driving protein-protein interactions. High-field, spin-label EPR at 95 GHz, i.e., 10 times higher than conventional EPR, is an upcoming technique to determine polarity parameters of the inside of proteins. Here we show that by 275 GHz EPR even the small polarity differences of sites at the protein surface can be discriminated. To do so, four single cysteine mutations were introduced at surface sites (positions 12, 27, 42, and 118) of azurin and spin labeled. By 275 GHz EPR in frozen solution, polarity/proticity differences between all four sites can be resolved, which is impossible by 95 GHz EPR. In addition, by 275 GHz EPR, two spectral components are observed for all mutants. The difference between them corresponds to one additional hydrogen bond.