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Colouring cryo-cooled crystals: online microspectrophotometry.

McGeehan J, Ravelli RB, Murray JW, Owen RL, Cipriani F, McSweeney S, Weik M, Garman EF - J Synchrotron Radiat (2009)

Bottom Line: The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K.The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals.The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.

View Article: PubMed Central - HTML - PubMed

Affiliation: EMBL, 6 rue Jules Horowitz, 38042 Grenoble, France.

ABSTRACT
X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200-1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.

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Related in: MedlinePlus

Spectra from X-irradiated crystals of apoferritin, N9 neuraminidase, HEWL and bovine trypsin. All but apoferritin show a peak around 400 nm corresponding to the disulfide radical anion. The broad absorption of the apoferritin from 500 nm into the UV region is probably due to the presence of residual iron in the protein crystal. The dose per 1 s of beam irradiation was estimated to be 4 × 104 Gy, giving a total of 1.2 × 106 Gy for the 30 s of exposure to which these crystals were subjected.
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fig5: Spectra from X-irradiated crystals of apoferritin, N9 neuraminidase, HEWL and bovine trypsin. All but apoferritin show a peak around 400 nm corresponding to the disulfide radical anion. The broad absorption of the apoferritin from 500 nm into the UV region is probably due to the presence of residual iron in the protein crystal. The dose per 1 s of beam irradiation was estimated to be 4 × 104 Gy, giving a total of 1.2 × 106 Gy for the 30 s of exposure to which these crystals were subjected.

Mentions: A superposition of the spectra from irradiated crystals of these proteins is shown in Fig. 5 ▶ (bovine trypsin, lysozyme, N9, ferritin); the absorption owing to the 400 nm disulphide radical anion is clearly visible. As for the glycerol series shown in Fig. 2 ▶, the absorption peaks reach a maximum within the time frame of the experiment, and then the final peak appears to decay by first-order kinetics.


Colouring cryo-cooled crystals: online microspectrophotometry.

McGeehan J, Ravelli RB, Murray JW, Owen RL, Cipriani F, McSweeney S, Weik M, Garman EF - J Synchrotron Radiat (2009)

Spectra from X-irradiated crystals of apoferritin, N9 neuraminidase, HEWL and bovine trypsin. All but apoferritin show a peak around 400 nm corresponding to the disulfide radical anion. The broad absorption of the apoferritin from 500 nm into the UV region is probably due to the presence of residual iron in the protein crystal. The dose per 1 s of beam irradiation was estimated to be 4 × 104 Gy, giving a total of 1.2 × 106 Gy for the 30 s of exposure to which these crystals were subjected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651762&req=5

fig5: Spectra from X-irradiated crystals of apoferritin, N9 neuraminidase, HEWL and bovine trypsin. All but apoferritin show a peak around 400 nm corresponding to the disulfide radical anion. The broad absorption of the apoferritin from 500 nm into the UV region is probably due to the presence of residual iron in the protein crystal. The dose per 1 s of beam irradiation was estimated to be 4 × 104 Gy, giving a total of 1.2 × 106 Gy for the 30 s of exposure to which these crystals were subjected.
Mentions: A superposition of the spectra from irradiated crystals of these proteins is shown in Fig. 5 ▶ (bovine trypsin, lysozyme, N9, ferritin); the absorption owing to the 400 nm disulphide radical anion is clearly visible. As for the glycerol series shown in Fig. 2 ▶, the absorption peaks reach a maximum within the time frame of the experiment, and then the final peak appears to decay by first-order kinetics.

Bottom Line: The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K.The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals.The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.

View Article: PubMed Central - HTML - PubMed

Affiliation: EMBL, 6 rue Jules Horowitz, 38042 Grenoble, France.

ABSTRACT
X-rays can produce a high concentration of radicals within cryo-cooled macromolecular crystals. Some radicals have large extinction coefficients in the visible (VIS) range of the electromagnetic spectrum, and can be observed optically and spectrally. An online microspectrophotometer with high temporal resolution has been constructed that is capable of measuring UV/VIS absorption spectra (200-1100 nm) during X-ray data collection. The typical X-ray-induced blue colour that is characteristic of a wide range of cryo-conditions has been identified as trapped solvated electrons. Disulphide-containing proteins are shown to form disulphide radicals at millimolar concentrations, with absorption maxima around 400 nm. The solvated electrons and the disulphide radicals seem to have a lifetime in the range of seconds up to minutes at 100 K. The temperature dependence of the kinetics of X-ray-induced radical formation is different for the solvated electrons compared with the disulphide radicals. The online microspectrophotometer provides a technique complementary to X-ray diffraction for analysing and characterizing intermediates and redox states of proteins and enzymes.

Show MeSH
Related in: MedlinePlus