Raman-markers of X-ray radiation damage of proteins

Despite their high relevance, the mechanisms of X-ray radiation damage on protein structure yet have to be completely established. Here, we used Raman microspectrophotometry to follow X-ray-induced chemical modifications on the structure of the model protein bovine pancreatic ribonuclease (RNase A). The combination of dose-dependent Raman spectra and ultrahigh resolution (eight structures solved using data collected between 0.85 and 1.17 Å resolution on the same single crystal) allowed direct observation of several radiation damage events, including covalent bond breakages and formation of radicals. Our results are relevant for analytical photodamage detection and provide implications for a detailed understanding of the mechanisms of photoproduct formation.