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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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Related in: MedlinePlus

Isomorphous difference density maps Fo,n − Fo,1 (where n is the data-set number) around the four disulfide bonds present in lysozyme. Maps are shown for Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy). Disulfide bonds are highlighted in yellow. Maps are contoured at +3σ (green) and −3σ (red). For Cys6–Cys127 the topmost part of the bond is Cys6, with the bottom being Cys127. The remaining bonds are positioned such that the label matches the residue positions in each figure, with the first to the left and the second to the right. Note that the dose indicated is the cumulative dose.
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fig6: Isomorphous difference density maps Fo,n − Fo,1 (where n is the data-set number) around the four disulfide bonds present in lysozyme. Maps are shown for Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy). Disulfide bonds are highlighted in yellow. Maps are contoured at +3σ (green) and −3σ (red). For Cys6–Cys127 the topmost part of the bond is Cys6, with the bottom being Cys127. The remaining bonds are positioned such that the label matches the residue positions in each figure, with the first to the left and the second to the right. Note that the dose indicated is the cumulative dose.

Mentions: Fo,n − Fo,1 maps contoured at 3σ for the disulfide bonds at each successive whole data-set dose are shown in Fig. 6 ▶. Positive density (green) indicates the presence of more electron density than seen in the 0.07 MGy data set, while negative density (dark red) results when the opposite is true. An inspection of the successive-dose electron-density maps in the area associated with each disulfide bond showed bond-specific effects.


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,n − Fo,1 (where n is the data-set number) around the four disulfide bonds present in lysozyme. Maps are shown for Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy). Disulfide bonds are highlighted in yellow. Maps are contoured at +3σ (green) and −3σ (red). For Cys6–Cys127 the topmost part of the bond is Cys6, with the bottom being Cys127. The remaining bonds are positioned such that the label matches the residue positions in each figure, with the first to the left and the second to the right. Note that the dose indicated is the cumulative dose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Isomorphous difference density maps Fo,n − Fo,1 (where n is the data-set number) around the four disulfide bonds present in lysozyme. Maps are shown for Fo,2 − Fo,1 (0.14 MGy), Fo,9 − Fo,1 (0.63 MGy) and Fo,15 − Fo,1 (1.05 MGy). Disulfide bonds are highlighted in yellow. Maps are contoured at +3σ (green) and −3σ (red). For Cys6–Cys127 the topmost part of the bond is Cys6, with the bottom being Cys127. The remaining bonds are positioned such that the label matches the residue positions in each figure, with the first to the left and the second to the right. Note that the dose indicated is the cumulative dose.
Mentions: Fo,n − Fo,1 maps contoured at 3σ for the disulfide bonds at each successive whole data-set dose are shown in Fig. 6 ▶. Positive density (green) indicates the presence of more electron density than seen in the 0.07 MGy data set, while negative density (dark red) results when the opposite is true. An inspection of the successive-dose electron-density maps in the area associated with each disulfide bond showed bond-specific effects.

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