XANES measurements of the rate of radiation damage to selenomethionine side chains

Abstract

The radiation-induced disordering of selenomethionine (SeMet) side chains represents a significant impediment to protein structure solution. Not only does the increased B-factor of these sites result in a serious drop in phasing power, but some sites decay much faster than others in the same unit cell. These radio-labile SeMet side chains decay faster than high-order diffraction spots with dose, making it difficult to detect this kind of damage by inspection of the diffraction pattern. The selenium X-ray absorbance near-edge spectrum (XANES) from samples containing SeMet was found to change significantly after application of X-ray doses of 10-100 MGy. Most notably, the sharp ;white line' feature near the canonical Se edge disappears. The change was attributed to breakage of the Cgamma-Se bond in SeMet. This spectral change was used as a probe to measure the decay rate of SeMet with X-ray dose in cryo-cooled samples. Two protein crystal types and 15 solutions containing free SeMet amino acid were examined. The damage rate was influenced by the chemical and physical condition of the sample, and the half-decaying dose for the selenium XANES signal ranged from 5 to 43 MGy. These decay rates were 34- to 3.8-fold higher than the rate at which the Se atoms interacted directly with X-ray photons, so the damage mechanism must be a secondary effect. Samples that cooled to a more crystalline state generally decayed faster than samples that cooled to an amorphous solid. The single exception was a protein crystal where a nanocrystalline cryoprotectant had a protective effect. Lowering the pH, especially with ascorbic or nitric acids, had a protective effect, and SeMet lifetime increased monotonically with decreasing sample temperature (down to 93 K). The SeMet lifetime in one protein crystal was the same as that of the free amino acid, and the longest SeMet lifetime measured was found in the other protein crystal type. This protection was found to arise from the folded structure of the protein molecule. A mechanism to explain observed decay rates involving the damaging species following the electric field lines around protein molecules is proposed.

Figure 1

XANES spectra from a 25 mM aqueous solution of SeMet in 25% glycerol at 93 K. These spectra indicate the measured photoabsorption cross section of Se to photons of the indicated energy. The solid green spectrum was taken before the sample absorbed any significant dose (< 1 MGy). The dashed brown line is a repeat of the same spectrum after the sample had absorbed a large dose (140 MGy). It is clear from these spectra that absorbed dose has an impact on XANES. The most prevalent change appears to be the loss of the so-called ‘white line’ peak absorbance. The height of the ‘white line’ peak (arrow) was used as the probe for the loss of the Se—C covalent bond.

Figure 2

Normalized plot of the fraction of unconverted SeMet (red error bars) against the X-ray fluence experienced by the sample at 12680 eV (droplet 38 from Table 1). The dose absorbed by the sample is indicated on the top x-axis and the fluence delivered is on the bottom x-axis. The blue solid line is the best-fit exponential curve to the data. The dotted brown line represents the fraction of selenium atoms in the sample that have experienced one or more direct collisions with an X-ray photon (photoionization events). Here we use a photoionization cross section of 157 cm² g^–1 for Se at 12680 eV (Hubbell, 1982). Clearly, the change in the XANES spectrum is too fast to be a direct result of primary photoionization of SeMet by X-ray photons. The breakdown of the Se—C bond must be the result of secondary or tertiary events.

Figure 3

Protection factor of nitrate ions. The fractional progression of the damage suffered by a solution of 25 mM SeMet in 25% (v/v) glycerol with either 1 M acetic acid (red error bars) or 1 M nitric acid (blue error bars) and the best-fit exponential curves to those data (solid grey and dotted black lines, respectively). The dose at which half of the SeMet has been damaged is called the ‘half-dose’ ($\mathrm{D}\frac{1}{2}$) for that sample. The difference between the $\mathrm{D}\frac{1}{2}$ of the nitric and acetic acid solutions divided by the $\mathrm{D}\frac{1}{2}$ of the acetic acid sample is called the ‘protection factor’, and reflects the degree of the positive impact nitrate ions have on the $\mathrm{D}\frac{1}{2}$ of SeMet. Errors in the $\mathrm{D}\frac{1}{2}$ determinations are propagated to evaluate whether the protection factor is significant.

Figure 4

This interrupted experiment demonstrates a lack of ‘dark progression’ of SeMet breakdown. Individual measurements of the peak ‘white line’ absorbance cross section are plotted against the time elapsed since the beginning of the experiment. The depicted run was interrupted after 5 h and the sample was returned to liquid nitrogen. After a 5 h delay, the sample was mounted, re-centered, and the rest of the decay curve was measured. There is no apparent change in the height of the white line across the 5 h during which the experiment was interrupted. When these data are plotted against the absorbed dose instead of time, they follow an exponential decay (Fig. 2).

Figure 5

Some experimental parameters protect SeMet from radiation damage. The ‘protection factor’ is the fractional increase in the ‘half-dose’ ($\mathrm{D}\frac{1}{2}$)of SeMet when the indicated ‘protective measure’ is used. The $\mathrm{D}\frac{1}{2}$ is the X-ray dose that will damage half of the SeMet in the sample. The ‘not ice’ column shows the protection factor of rapidly cooling a solution of SeMet to form an amorphous glassy solid as opposed to cooling it slowly to form ice Ih (droplet 110 in Table 1). The ‘not nano-ice’ column shows the protection factor of the same amorphous condition over the same solution cooled slightly slower to form the nano-crystalline solid discussed in the text (droplet 130). ‘Low pH’ is the protection factor of preparing the amorphous sample with 1 M acetic acid added to the solution over adding 0.6 M NaOH (droplets 39 and 52). ‘Ascorbate’ is the protection factor of using 1 M ascorbic acid instead of acetic acid (droplets 20 and 21). ‘Nitrate’ is the protection factor of using 1 M nitric acid instead of acetic acid (droplets 25 and 21). ‘Low temperature’ is the protection factor of running the experiment at 93 K instead of 140 K in 0.6 M NaOH (droplets 86 and 88). All other experiments in this figure were run at 93 K. See Fig. 6 for details. ‘In peptide’ is the protection factor of unfolded GCN4-N16A-p1 peptide at 20 mM (80 g L^–1) boiled in 9.5 M urea over free SeMet at 25 mM also boiled in 9.5 M urea with 80 g L^–1 insulin. ‘Folded’ is the protection factor of folded GCN4-N16A-p1 peptide in solution (no urea) over the same concentration of unfolded peptide (boiled in 9.5 M urea). ‘Crystallized’ is the protection factor for GCN4-N16A-p1 peptide crystals over the folded peptide in solution. ‘GCN4 xtal’ is the protection factor of a crystal of the GCN4-N16A-p1 peptide over free SeMet in the crystal cryoprotectant. This combines the previous three protective measures. ‘Not NE1 xtal’ is the protection factor of a crystal of free SeMet in solution over the SeMet side chains in the E1 domain of NEDD8, but note that not all crystals are protective. ‘Ice vs GCN4’ is the protection factor of the largest observed $\mathrm{D}\frac{1}{2}$ (43.2 MGy) over the smallest observed $\mathrm{D}\frac{1}{2}$ (5 MGy). This final protection factor is 750% and dwarfs all the others, so it is indicated by a number over the graph. Although individual protection factors are small enough to be difficult to measure, the effects were generally additive and the range of observed $\mathrm{D}\frac{1}{2}$ values for SeMet does vary widely.

Figure 6

Temperature dependence of the $\mathrm{D}\frac{1}{2}$ of 31 mM SeMet in 0.6 M NaOH and 25% (v/v) glycerol. $\mathrm{D}\frac{1}{2}$ values for samples of this solution were measured with the sample cooler set for different temperatures (error bars). Lower temperatures continued to increase the $\mathrm{D}\frac{1}{2}$ of SeMet down to ~92 K which was the lowest temperature obtainable with this equipment. The solid line is a smooth curve (to guide the eye). There is a clear transition in the slope of the temperature dependence of the $\mathrm{D}\frac{1}{2}$ around 130 K which may correspond to the glass transition of pure water near this same temperature, allowing new factors to come into play above 130 K.