X-RAY INDUCED PHOTODAMAGE IN PROTEINS: A RAMAN-ASSISTED BIOCRYSTALLOGRAPHIC STUDY

Currently, few sites provide the possibility to perform simultaneous Raman/X-ray diffraction of single macromolecular crystals. In these recent Raman-assisted crystallography applications, Raman microscopy has been representing a fine servant of a dominant X-ray Crystallography, and its highest scope was the reinforcement of structural data [1, 2]. The strategy of our work is to overturn this paradigm, focusing Crystallography-assisted Raman on the rich spectroscopic data that are well transferable to many bio-analytical applications. A Synchrotron with available Raman microscope, SLS [3], has been used to collect high-resolution crystallographic data on unusual states of model biomolecules. X-ray induced radiation damage is a frequent phenomenon, especially when using third generation synchrotrons. Raman microscopy prior and after X-ray data collection can be a valuable tool to detect artifacts derived from radiation damage. A case of study is presented: ultra high resolution crystal structure (0.8 Å) of ribonuclease A at different X- ray doses. Herein, we provide ultra high resolution data (0.8 Å resolution) at six different doses (from 0,4 up to 24 MGy), with a clear disappearance of Raman bands due to photodamage. We observe, studying RNase A crystals, novel Raman bands (photoproducts) appearing upon X-ray dose increases. Ultra-high resolutions provide electron density maps of such compounds derived from degradation of methionine, aspartic acids and tyrosine side chains. Some of these appearing bands have been identified, whereas other Raman markers are still under investigation, combining Raman data with ultra-high resolution crystallography. Ultra-high resolutions have provided electron density maps of compounds derived from degradation of methionine and tyrosines. Some of these appearing bands have been identified, e.g. methyl radicals at 1356 cm-1 . Other Raman markers are still under investigation (987 cm-1).