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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 for radical trapping in lysozyme crystals irradiated with 70 keV X-rays at 4 K. Data for the concentration of total trapped radicals, R(tot), are shown for three different crystals using open black symbols (left y axis). Data for radicals formed by reduction of RSSR, R(SS), are shown using closed blue symbols (right y axis). See text regarding the normalization of the data using the measured mass of crystal 1. The curves were obtained by a nonlinear least-squares fit of the data using equations (6) and (7) with the parameters detailed in the figure.
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fig5: The dose response for radical trapping in lysozyme crystals irradiated with 70 keV X-rays at 4 K. Data for the concentration of total trapped radicals, R(tot), are shown for three different crystals using open black symbols (left y axis). Data for radicals formed by reduction of RSSR, R(SS), are shown using closed blue symbols (right y axis). See text regarding the normalization of the data using the measured mass of crystal 1. The curves were obtained by a nonlinear least-squares fit of the data using equations (6) and (7) with the parameters detailed in the figure.

Mentions: In Fig. 4 ▶, EPR spectra are shown for four different X-ray doses. At low doses in the EPR experiment, e.g. between 10 and 20 kGy, the spectrum intensity increases linearly with dose. At higher doses, e.g. 200–400 kGy, a plateau is reached. The blue traces in Fig. 4 ▶ are simulations of the component, which as described above is associated with the low-field signal assigned exclusively to . The double integral of the experimental and calculated spectra gave the radical concentrations R(tot) and R(SS), respectively. These concentrations were used in the dose-response curves shown in Fig. 5 ▶. In Fig. 4 ▶ the peak from the growing component is indicated along with a peak from trace amounts of Mn+ known to be present in the experimental setup.


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 for radical trapping in lysozyme crystals irradiated with 70 keV X-rays at 4 K. Data for the concentration of total trapped radicals, R(tot), are shown for three different crystals using open black symbols (left y axis). Data for radicals formed by reduction of RSSR, R(SS), are shown using closed blue symbols (right y axis). See text regarding the normalization of the data using the measured mass of crystal 1. The curves were obtained by a nonlinear least-squares fit of the data using equations (6) and (7) with the parameters detailed in the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: The dose response for radical trapping in lysozyme crystals irradiated with 70 keV X-rays at 4 K. Data for the concentration of total trapped radicals, R(tot), are shown for three different crystals using open black symbols (left y axis). Data for radicals formed by reduction of RSSR, R(SS), are shown using closed blue symbols (right y axis). See text regarding the normalization of the data using the measured mass of crystal 1. The curves were obtained by a nonlinear least-squares fit of the data using equations (6) and (7) with the parameters detailed in the figure.
Mentions: In Fig. 4 ▶, EPR spectra are shown for four different X-ray doses. At low doses in the EPR experiment, e.g. between 10 and 20 kGy, the spectrum intensity increases linearly with dose. At higher doses, e.g. 200–400 kGy, a plateau is reached. The blue traces in Fig. 4 ▶ are simulations of the component, which as described above is associated with the low-field signal assigned exclusively to . The double integral of the experimental and calculated spectra gave the radical concentrations R(tot) and R(SS), respectively. These concentrations were used in the dose-response curves shown in Fig. 5 ▶. In Fig. 4 ▶ the peak from the growing component is indicated along with a peak from trace amounts of Mn+ known to be present in the experimental setup.

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