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.

Figure 1

Room-temperature data for relative B factor versus dose for lysozyme crystals soaked in six representative small-molecule compounds that had neither protecting nor sensitizing effects. See Table 1 ▶ for a full list of the compounds studied.

Figure 2

Room-temperature data for relative B factor versus dose for five native lysozyme crystals irradiated by a 10 keV X-ray beam at different dose rates. No variation of slope with dose rate is observed for dose rates differing by as much as 16; only the total dose is relevant. For the 11.0 kGy s⁻¹ data set (the four circle-based symbols), the beam was turned off for 10 h between the first and second data points and between the third and fourth data points. No dark progression is observed: damage picks up where it left off prior to the break. The slight increase in B after the break arises from the small dose received in acquiring each data point.

Figure 3

(a) Relative B factor versus dose for lysozyme crystals soaked in sodium nitrate and for two native crystals at room temperature. Experimental uncertainties are reflected in the difference between the native crystals. The solid lines are guides to the eye, indicating the overall trend of damage rates without and with nitrate. (b) Relative B factor versus dose for nitrate-soaked thaumatin crystals at room temperature.

Figure 4

Fourier difference maps comparing native tetragonal lysozyme crystals and crystals soaked in 100 mM nitrate. Top-row structures were acquired at T = 100 K and bottom-row structures at T = 295 K. Left column: difference maps (F _nitrate,obs − F _native,obs, ϕ_native) acquired on ‘fresh’ crystals prior to delivery of a large dose. Purple contours correspond to levels of +4.4σ and +3.4σ at 100 and 295 K, respectively. At T = 100 K, two water molecules and the chloride have moved to new positions. At T = 295 K, the loop with Thr69 relocated to a new position. Right column: difference maps (F _fresh,obs − F _dosed,obs, ϕ_fresh) comparing ‘fresh’ and ‘dosed’ crystals, where damage arises from a large dose (0.12 MGy at room temperature and 1.9 MGy at T = 100 K) delivered between structural data sets. Red contours indicate damage to groups in native (nitrate-free) crystals, with levels of +4.3σ and +3.1σ at 100 and 295 K, respectively; yellow contours show damage in nitrate-soaked crystals, with levels of +4.7σ and +3.2σ at 100 and 295 K, respectively. At room temperature, the sulfur bridge Cys6–Cys127 appears to be protected by nitrate.