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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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Isomorphous difference density maps Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy) for residues Met12 and Met105. Maps are contoured at 3σ in green and −3σ in dark red.
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fig7: Isomorphous difference density maps Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy) for residues Met12 and Met105. Maps are contoured at 3σ in green and −3σ in dark red.

Mentions: In addition to the S atoms in the four disulfide bonds, there are also two additional S atoms present in lysozyme in Met12 and Met105. Both of these have zero solvent accessibility. The Fo,n − Fo,1 maps for these residues are shown in Fig. 7 ▶. For Met12 there is little if any indication of dose-related damage and negligible initial damage of the carbon–sulfur bond in Met105. This possible localized damage on the S atom is present in the Fo,n − Fo,1 maps from the initial map to Fo,6 − Fo,1 (0.49 MGy). However, after the sixth data set it is no longer localized. Overall, the effect appears to be marginal.


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)

Isomorphous difference density maps Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy) for residues Met12 and Met105. Maps are contoured at 3σ in green and −3σ in dark red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Isomorphous difference density maps Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy) for residues Met12 and Met105. Maps are contoured at 3σ in green and −3σ in dark red.
Mentions: In addition to the S atoms in the four disulfide bonds, there are also two additional S atoms present in lysozyme in Met12 and Met105. Both of these have zero solvent accessibility. The Fo,n − Fo,1 maps for these residues are shown in Fig. 7 ▶. For Met12 there is little if any indication of dose-related damage and negligible initial damage of the carbon–sulfur bond in Met105. This possible localized damage on the S atom is present in the Fo,n − Fo,1 maps from the initial map to Fo,6 − Fo,1 (0.49 MGy). However, after the sixth data set it is no longer localized. Overall, the effect appears to be marginal.

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