Can radiation damage to protein crystals be reduced using small-molecule compounds?

Abstract

Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T=100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

Spatial distribution of radiation damage to crystalline proteins at 25–300 K

“…The B-factor-derived dose estimates given above should thus be considered accurate to within 50%. However, unlike in our quantitative studies of global radiation damage (Kmetko et al, 2006(Kmetko et al, , 2011Warkentin & Thorne, 2010;Warkentin et al, 2011Warkentin et al, , 2012, none of the present conclusions rely on the precise global damage or dose estimates and are robust against sample-to-sample variability of global sensitivity.…”

“…Protein crystals are approximately 10-130 times more sensitive to global damage at room temperature than at cryogenic temperatures ($100 K; Blake & Phillips, 1962;Teng & Moffat, 2002;Kmetko et al, 2006Kmetko et al, , 2011Southworth-Davies et al, 2007;Barker et al, 2009;Warkentin & Thorne, 2010). Solvent and atomic radical diffusion and diffusive motions of protein side chains and larger structural elements are suppressed below the proteinsolvent glass-transition temperature near T ' 200 K and become negligible by $100 K (Rodgers, 1994;Garman & Schneider, 1997;Weik, Kryger et al, 2001;Garman, 2003;Weik et al, 2004Weik et al, , 2005Warkentin & Thorne, 2009, 2010.…”

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

“…However, the advantages of including such compounds are unclear. There are several studies which report conflicting evidence of their efficacy at mitigating radiation damage [30][31][32][33], and only one study has proved it beneficial during room-temperature data collection [34]. In the worst-case scenario, inclusion of metal containing scavengers and crystallisation reagents will merely add to the heavy atom content of the crystal and increase its X-ray absorption, making it more sensitive to radiation damage.…”

Radiation chemists look at damage in redox proteins induced by X‐rays

“…Still, a study of lysozyme crystals showed that none of the 19 different added molecules caused a decrease in global protein damage at 100 K while many acted as sensitizers at room temperature. This was attributed to having the additives present some distance from the site of primary X‐ray absorption by the protein . Hence, as detailed below, the main outcome of the absorbed radiation is production of mobile species such as reducing and oxidizing radicals.…”