Evidence for the formation of disulfide radicals in protein crystals upon X-ray irradiation

Abstract

Irradiation of proteins with intense X-ray radiation produced by third-generation synchrotron sources generates specific structural and chemical alterations, including breakage of disulfide bonds and decarboxylation. In this paper, disulfide bond lengths in irradiated crystals of the enzyme Torpedo californica acetylcholinesterase are examined based on quantum simulations and on experimental data published previously. The experimental data suggest that one disulfide bond elongates by approximately 0.7 A upon X-ray irradiation as seen in a series of nine data sets collected on a single crystal. Simulation of the same bond suggests elongation by a similar value if a disulfide-radical anion is formed by trapping an electron. The absorption spectrum of a crystal irradiated under similar conditions shows a peak at approximately 400 nm, which in aqueous solution has been attributed to disulfide radicals. The results suggest that the formation of disulfide radicals in protein crystals owing to X-ray irradiation can be observed experimentally, both by structural means and by absorption spectroscopy.

Colouring cryo-cooled crystals: online microspectrophotometry

“…Using the offline spectrophotometer installed at the ESRF cryobench (Bourgeois et al, 2002), we observed that irradiated crystals of lysozyme and of acetylcholinesterase had an absorption peak at around 400 nm (Murray & Garman, 2002;Weik et al, 2002). From previously reported work (Favaudon et al, 1990), this was identified as the disulfide radical anion (RSSR À ) that is elongated by 0.7 Å with respect to a neutral disulfide bond (Weik et al, 2002).…”

“…We initiated its development after cumbersome experiments (Murray & Garman, 2002;Weik et al, 2002) where crystals were transferred back and forth between the beamline and an offline microspectrophotometer (Bourgeois et al, 2002). Mirror lenses were custom designed to allow the incorporation of a spectrophotometer into the crowded space around the sample holder.…”

Measurement errors and their consequences in protein crystallography

“…Another type of non-isomorphism problem is caused by large doses of radiation. Some chemical groups are affected more readily than others (Burmeister, 2000;Weik et al, 2000Weik et al, , 2002Leiros et al, 2001). In addition, molecules can rotate and translate in the crystal lattice.…”

Modelling and refining site-specific radiation damage in SAD/MAD phasing

“…where the summation is (in the asymmetric unit of the crystal cell) over all atoms that are initially (before the irradiation) present in the substructure and d h, j is the dose at the jth measurement of reflection h. In many cases, site-specific radiation damage consists of the breaking of chemical bonds, followed by the apparent 'disappearance' of atoms that were engaged in these bonds (Burmeister, 2000;Weik et al, 2000Weik et al, , 2002. The atoms do not necessarily disappear from the sample, but they become disordered and are thus no longer contributing to the diffraction signal.…”

“…Radiation damage first occurs at discrete well localized sites (Burmeister, 2000;Weik et al, 2000Weik et al, , 2002Ennifar et al, 2002). Only in a second phase does it affect the whole of the crystal structure, causing longrange disorder with its characteristic consequences of overall intensity decay and loss of high-resolution reflections.…”