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Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.

Sutton KA, Black PJ, Mercer KR, Garman EF, Owen RL, Snell EH, Bernhard WA - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied.The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized.Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14086, USA.

ABSTRACT
Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

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The dose-response behavior of the  radical in a lysozyme crystal observed through UV–Vis absorption spectroscopy. (a) The spectra show the rapid rise in the overall signal owing to the increase in radical concentration and the temporal evolution of absorption peaks at 400 and 580 nm. An isosbestic point is present at 480 nm. (b) Absorbance at 400 nm ( radical signal) as a function of absorbed dose with single- and double-exponential fits overlaid (residuals are shown in the Supplementary Material). The crystal was subjected to a total absorbed dose of ∼5 MGy (∼80 s at 61.8 kGy s−1) before the shutter was closed (see text for details). (c) Variation in fit parameters as a function of dose rate; no systematic trend is observed.
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fig2: The dose-response behavior of the radical in a lysozyme crystal observed through UV–Vis absorption spectroscopy. (a) The spectra show the rapid rise in the overall signal owing to the increase in radical concentration and the temporal evolution of absorption peaks at 400 and 580 nm. An isosbestic point is present at 480 nm. (b) Absorbance at 400 nm ( radical signal) as a function of absorbed dose with single- and double-exponential fits overlaid (residuals are shown in the Supplementary Material). The crystal was subjected to a total absorbed dose of ∼5 MGy (∼80 s at 61.8 kGy s−1) before the shutter was closed (see text for details). (c) Variation in fit parameters as a function of dose rate; no systematic trend is observed.

Mentions: X-ray-induced changes in the optical absorption of lysozyme crystals upon irradiation were monitored using an online microspectrophotometer as described above. The increased absorbance at 400 nm is attributable to the radical species (Weik et al., 2002 ▶; Southworth-Davies & Garman, 2007 ▶) and an increase in absorbance at this wavelength was clearly observed in all samples. This was accompanied by a peak in absorption at ∼580 nm (Fig. 2 ▶a) which is attributable to the formation of solvated electrons (McGeehan et al., 2009 ▶). Both of these features can clearly be seen in the spectral series in Fig. 2 ▶(a), which shows the results of a continuous 80 s irradiation with a cumulative dose of 5 MGy (dose rate of 62 kGy s−1). The absorbance at 400 nm increases rapidly before saturating and the 580 nm peak owing to solvated electrons has an observed maximum at the earliest recorded point. This peak may have been higher at earlier time points (below 200 ms) that were not captured in the experiment. The observation that this solvated electron signal (580 nm) decreases as the 400 nm absorption peak increases supports our model; the solvated electrons are depleted as and other one-electron reduction products are formed. This is in agreement with a related study on lysozyme by Allan et al. (2013 ▶) also using UV–visible microspectrophotometry. Allen and coworkers observed an initial rise in the 580 nm absorption with increasing dose, followed by a fall in this signal corresponding to an increase in absorption at 400 nm. In Fig. 2 ▶(b)1 the dose-dependent increase in absorbance at 400 nm is plotted. The dose-response curves were fitted to both a single- and a double-exponential function Abs = A0 + B1exp(D/d1) + B2exp(D/d2), where A0 is the baseline, B1, B2, d1 and d2 are constants and D is the dose. For the double-exponential fit d1 and d2 were defined such that d1 > d2. B2 was defined as zero for the single-exponential fit. All data could be well fitted with a single or double exponential with an R2 of ≥0.95, although visual inspection of the fits showed that the double-exponential parameterization better describes the data (Fig. 2 ▶b). The constants d1 and d2 are shown as a function of dose rate in Fig. 2 ▶(c) for both single- and double-exponential fits. We define the saturating dose, D90, as the point at which the absorbance reaches 90% of the maximum above baseline. This is the dose at which fast changes no longer dominate. In this case the D90 for lysozyme crystals averages 0.51–0.77 MGy (depending on the single- or double-exponential fit), but the variability is large (see Table 2 ▶). There was no clear indication of dose-rate dependence on the saturation level.


Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.

Sutton KA, Black PJ, Mercer KR, Garman EF, Owen RL, Snell EH, Bernhard WA - Acta Crystallogr. D Biol. Crystallogr. (2013)

The dose-response behavior of the  radical in a lysozyme crystal observed through UV–Vis absorption spectroscopy. (a) The spectra show the rapid rise in the overall signal owing to the increase in radical concentration and the temporal evolution of absorption peaks at 400 and 580 nm. An isosbestic point is present at 480 nm. (b) Absorbance at 400 nm ( radical signal) as a function of absorbed dose with single- and double-exponential fits overlaid (residuals are shown in the Supplementary Material). The crystal was subjected to a total absorbed dose of ∼5 MGy (∼80 s at 61.8 kGy s−1) before the shutter was closed (see text for details). (c) Variation in fit parameters as a function of dose rate; no systematic trend is observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3852651&req=5

fig2: The dose-response behavior of the radical in a lysozyme crystal observed through UV–Vis absorption spectroscopy. (a) The spectra show the rapid rise in the overall signal owing to the increase in radical concentration and the temporal evolution of absorption peaks at 400 and 580 nm. An isosbestic point is present at 480 nm. (b) Absorbance at 400 nm ( radical signal) as a function of absorbed dose with single- and double-exponential fits overlaid (residuals are shown in the Supplementary Material). The crystal was subjected to a total absorbed dose of ∼5 MGy (∼80 s at 61.8 kGy s−1) before the shutter was closed (see text for details). (c) Variation in fit parameters as a function of dose rate; no systematic trend is observed.
Mentions: X-ray-induced changes in the optical absorption of lysozyme crystals upon irradiation were monitored using an online microspectrophotometer as described above. The increased absorbance at 400 nm is attributable to the radical species (Weik et al., 2002 ▶; Southworth-Davies & Garman, 2007 ▶) and an increase in absorbance at this wavelength was clearly observed in all samples. This was accompanied by a peak in absorption at ∼580 nm (Fig. 2 ▶a) which is attributable to the formation of solvated electrons (McGeehan et al., 2009 ▶). Both of these features can clearly be seen in the spectral series in Fig. 2 ▶(a), which shows the results of a continuous 80 s irradiation with a cumulative dose of 5 MGy (dose rate of 62 kGy s−1). The absorbance at 400 nm increases rapidly before saturating and the 580 nm peak owing to solvated electrons has an observed maximum at the earliest recorded point. This peak may have been higher at earlier time points (below 200 ms) that were not captured in the experiment. The observation that this solvated electron signal (580 nm) decreases as the 400 nm absorption peak increases supports our model; the solvated electrons are depleted as and other one-electron reduction products are formed. This is in agreement with a related study on lysozyme by Allan et al. (2013 ▶) also using UV–visible microspectrophotometry. Allen and coworkers observed an initial rise in the 580 nm absorption with increasing dose, followed by a fall in this signal corresponding to an increase in absorption at 400 nm. In Fig. 2 ▶(b)1 the dose-dependent increase in absorbance at 400 nm is plotted. The dose-response curves were fitted to both a single- and a double-exponential function Abs = A0 + B1exp(D/d1) + B2exp(D/d2), where A0 is the baseline, B1, B2, d1 and d2 are constants and D is the dose. For the double-exponential fit d1 and d2 were defined such that d1 > d2. B2 was defined as zero for the single-exponential fit. All data could be well fitted with a single or double exponential with an R2 of ≥0.95, although visual inspection of the fits showed that the double-exponential parameterization better describes the data (Fig. 2 ▶b). The constants d1 and d2 are shown as a function of dose rate in Fig. 2 ▶(c) for both single- and double-exponential fits. We define the saturating dose, D90, as the point at which the absorbance reaches 90% of the maximum above baseline. This is the dose at which fast changes no longer dominate. In this case the D90 for lysozyme crystals averages 0.51–0.77 MGy (depending on the single- or double-exponential fit), but the variability is large (see Table 2 ▶). There was no clear indication of dose-rate dependence on the saturation level.

Bottom Line: The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied.The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized.Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14086, USA.

ABSTRACT
Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

Show MeSH
Related in: MedlinePlus