Colouring cryo-cooled crystals: online microspectrophotometry

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 cryoconditions has been identified as trapped solvated electrons. Disulphidecontaining 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.

Temperature-dependent macromolecular X-ray crystallography

“…that of hydrated electrons) and in the protein research papers (e.g. that of disulfide radicals) is temperature-independent as determined by UV-visible absorption spectroscopy performed online at a synchrotron beamline (McGeehan et al, 2009). The sudden drop in viscosity at the solvent glass transition (which takes place at 150 K or at a higher temperature) allows an increase in the mobility of radicals that were trapped in the amorphous solvent at lower temperatures and therefore the radical lifetime decreases.…”

Radiation damage in protein crystals examined under various conditions by different methods

“…When trapped in water, the hydrated (or more generally solvated) electrons give a characteristic broad optical absorption spectrum (Ershov & Pikaev, 1968). This spectrum of the hydrated electron builds up during the X-ray exposure but undergoes partial decay (McGeehan et al, 2009) when the beam is switched off as some electrons recombine (e.g. with the various positively charged holes).…”

“…Hough et al, 2008). In this issue, papers by McGeehan et al (2009) and Owen, Pearson et al (2009) describe experimental arrangements which enable routine measurements of this kind, together with (resonance) Raman spectroscopy. EXAFS can be used to measure metal ligand distances to an accuracy of 0.02 Å and is therefore sensitive to small movements of atoms surrounding a metal atom.…”

“…Many of these changes can be observed by a variety of spectroscopic techniques [e.g. UV/Vis spectroscopy (Beitlich et al, 2007;McGeehan et al, 2009), Raman (Carpentier et al, 2007), XAS (Corbett et al, 2007) and EPR (Utschig et al, 2008)]. When trapped in water, the hydrated (or more generally solvated) electrons give a characteristic broad optical absorption spectrum (Ershov & Pikaev, 1968).…”

Imaging local electric fields produced upon synchrotron X-ray exposure

“…Specific damage occurs when irradiation causes specific chemical changes within the protein such as loss of side chains or disulfide bridge linkages (2,3), damage to active sites (2), and damage to metal centers within the protein (1). Disulfide anion radicals, evidence of specific damage, have previously been observed by UV-vis microspectrophotometry after X-ray exposure for tens of milliseconds (4). Damage to nonspecific sites, known also as global damage, results in a decrease in the intensity of the high-angle spots with increasing X-ray dose, and therefore an overall loss in resolution (1,5).…”