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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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Absorbance measured for (a) 20 × 1 s burns and (b) a single 20 s burn. The absorbed dose per 1 s exposure was 287 kGy. The cumulative dose over 20 s was thus 5.74 MGy. (a) The multiple burns show a progressively smaller change in absorption for the same absorbed dose and rapid loss of  was observed after each pulse. (b) The single continuous 20 s burn highlights the post-exposure decay of the disulfide peak, which is best described by a two-rate model, in agreement with previous observations (Owen et al., 2011 ▶; Beitlich et al., 2007 ▶).
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fig3: Absorbance measured for (a) 20 × 1 s burns and (b) a single 20 s burn. The absorbed dose per 1 s exposure was 287 kGy. The cumulative dose over 20 s was thus 5.74 MGy. (a) The multiple burns show a progressively smaller change in absorption for the same absorbed dose and rapid loss of was observed after each pulse. (b) The single continuous 20 s burn highlights the post-exposure decay of the disulfide peak, which is best described by a two-rate model, in agreement with previous observations (Owen et al., 2011 ▶; Beitlich et al., 2007 ▶).

Mentions: The change in absorbance from a series of 1 s exposures interspersed with a 5 s rest period is shown in Fig. 3 ▶(a). Despite a rapid reduction in absorbance when the X-ray shutter was closed for the rest period, saturation at 400 nm was still achieved swiftly with a progressively smaller change in absorption for the same additional absorbed dose. The reduction in absorption seen during the rest period indicates that some fraction of was lost owing to recombination and/or deprotonation, but the dominating increase over time indicates that some fraction was stable at 100 K. The post-exposure decay of the disulfide peak at 400 nm subsequent to a 20 s continuous X-ray exposure is shown in Fig. 3 ▶(b). The decay follows a double-exponential form with rate constants d1 and d2 equal to 13.1 ± 1.6 and 140.2 ± 20.7 s−1, respectively (Fig. 3 ▶b). The fit of the decay by a double-exponential function is in agreement with previous observations (Owen et al., 2011 ▶; Beitlich et al., 2007 ▶). Both results, Figs. 3 ▶(a) and 3 ▶(b), add support to the multi-track model comprising both product formation and destruction.


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)

Absorbance measured for (a) 20 × 1 s burns and (b) a single 20 s burn. The absorbed dose per 1 s exposure was 287 kGy. The cumulative dose over 20 s was thus 5.74 MGy. (a) The multiple burns show a progressively smaller change in absorption for the same absorbed dose and rapid loss of  was observed after each pulse. (b) The single continuous 20 s burn highlights the post-exposure decay of the disulfide peak, which is best described by a two-rate model, in agreement with previous observations (Owen et al., 2011 ▶; Beitlich et al., 2007 ▶).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Absorbance measured for (a) 20 × 1 s burns and (b) a single 20 s burn. The absorbed dose per 1 s exposure was 287 kGy. The cumulative dose over 20 s was thus 5.74 MGy. (a) The multiple burns show a progressively smaller change in absorption for the same absorbed dose and rapid loss of was observed after each pulse. (b) The single continuous 20 s burn highlights the post-exposure decay of the disulfide peak, which is best described by a two-rate model, in agreement with previous observations (Owen et al., 2011 ▶; Beitlich et al., 2007 ▶).
Mentions: The change in absorbance from a series of 1 s exposures interspersed with a 5 s rest period is shown in Fig. 3 ▶(a). Despite a rapid reduction in absorbance when the X-ray shutter was closed for the rest period, saturation at 400 nm was still achieved swiftly with a progressively smaller change in absorption for the same additional absorbed dose. The reduction in absorption seen during the rest period indicates that some fraction of was lost owing to recombination and/or deprotonation, but the dominating increase over time indicates that some fraction was stable at 100 K. The post-exposure decay of the disulfide peak at 400 nm subsequent to a 20 s continuous X-ray exposure is shown in Fig. 3 ▶(b). The decay follows a double-exponential form with rate constants d1 and d2 equal to 13.1 ± 1.6 and 140.2 ± 20.7 s−1, respectively (Fig. 3 ▶b). The fit of the decay by a double-exponential function is in agreement with previous observations (Owen et al., 2011 ▶; Beitlich et al., 2007 ▶). Both results, Figs. 3 ▶(a) and 3 ▶(b), add support to the multi-track model comprising both product formation and destruction.

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