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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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Temperature-dependence of disulfide radical lifetimes. The optical density (OD) of an orthorhombic trypsin crystal irradiated for ten beam pulses of 1 s followed over time at 400 (red), 600 (blue) and 750 nm (green) at (a) 100, (b) 130 and (c) 160 K. The decay of the signals at 400 nm after the final pulses was fitted to a first-order exponential to extract disulfide-radical lifetimes. The absorbance at 600 nm corresponds to the presence of trapped electrons; the one at 750 nm is shown as a reference wavelength. The dose per 1 s of beam irradiation was estimated using RADDOSE (Murray et al., 2004 ▶) to be 4.4 × 104 Gy, giving a total of 4.4 × 105 Gy for the 10 s of data shown here.
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fig6: Temperature-dependence of disulfide radical lifetimes. The optical density (OD) of an orthorhombic trypsin crystal irradiated for ten beam pulses of 1 s followed over time at 400 (red), 600 (blue) and 750 nm (green) at (a) 100, (b) 130 and (c) 160 K. The decay of the signals at 400 nm after the final pulses was fitted to a first-order exponential to extract disulfide-radical lifetimes. The absorbance at 600 nm corresponds to the presence of trapped electrons; the one at 750 nm is shown as a reference wavelength. The dose per 1 s of beam irradiation was estimated using RADDOSE (Murray et al., 2004 ▶) to be 4.4 × 104 Gy, giving a total of 4.4 × 105 Gy for the 10 s of data shown here.

Mentions: The lifetime of disulfide radicals as a function of temperature was investigated with flash-cooled orthorhombic bovine trypsin crystals. Ten consecutive X-ray irradiations at 100 K produced disulfide radicals, of which the absorption at 400 nm was followed as a function of time (Fig. 6a ▶). Upon the opening of the shutter, the absorbance at 400 nm increased instantaneously. During the 1 s X-ray exposure, the absorbance increased further, owing to the dose-dependent accumulation of disulfide radicals. In the subsequent period of 4 s without irradiation, the absorbance does not remain constant but decays. In the following periods with and without X-ray pulses, the absorbance again increases and decreases, respectively. From the decay after the final (tenth) X-ray pulse, a half life of around 300 s was obtained. Experiments on a previously unexposed part of the same crystal at temperatures of 130 and 160 K showed approximately the same decay rate (Figs. 6b and 6c ▶).


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)

Temperature-dependence of disulfide radical lifetimes. The optical density (OD) of an orthorhombic trypsin crystal irradiated for ten beam pulses of 1 s followed over time at 400 (red), 600 (blue) and 750 nm (green) at (a) 100, (b) 130 and (c) 160 K. The decay of the signals at 400 nm after the final pulses was fitted to a first-order exponential to extract disulfide-radical lifetimes. The absorbance at 600 nm corresponds to the presence of trapped electrons; the one at 750 nm is shown as a reference wavelength. The dose per 1 s of beam irradiation was estimated using RADDOSE (Murray et al., 2004 ▶) to be 4.4 × 104 Gy, giving a total of 4.4 × 105 Gy for the 10 s of data shown here.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Temperature-dependence of disulfide radical lifetimes. The optical density (OD) of an orthorhombic trypsin crystal irradiated for ten beam pulses of 1 s followed over time at 400 (red), 600 (blue) and 750 nm (green) at (a) 100, (b) 130 and (c) 160 K. The decay of the signals at 400 nm after the final pulses was fitted to a first-order exponential to extract disulfide-radical lifetimes. The absorbance at 600 nm corresponds to the presence of trapped electrons; the one at 750 nm is shown as a reference wavelength. The dose per 1 s of beam irradiation was estimated using RADDOSE (Murray et al., 2004 ▶) to be 4.4 × 104 Gy, giving a total of 4.4 × 105 Gy for the 10 s of data shown here.
Mentions: The lifetime of disulfide radicals as a function of temperature was investigated with flash-cooled orthorhombic bovine trypsin crystals. Ten consecutive X-ray irradiations at 100 K produced disulfide radicals, of which the absorption at 400 nm was followed as a function of time (Fig. 6a ▶). Upon the opening of the shutter, the absorbance at 400 nm increased instantaneously. During the 1 s X-ray exposure, the absorbance increased further, owing to the dose-dependent accumulation of disulfide radicals. In the subsequent period of 4 s without irradiation, the absorbance does not remain constant but decays. In the following periods with and without X-ray pulses, the absorbance again increases and decreases, respectively. From the decay after the final (tenth) X-ray pulse, a half life of around 300 s was obtained. Experiments on a previously unexposed part of the same crystal at temperatures of 130 and 160 K showed approximately the same decay rate (Figs. 6b and 6c ▶).

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