Structural changes in a cryo-cooled protein crystal owing to radiation damage.

The high intensity of third-generation X-ray sources, along with the development of cryo-cooling of protein crystals at temperatures around 100 K, have made it possible to extend the diffraction limit of crystals and to reduce their size. However, even with cryo-cooled crystals, radiation damage becomes a limiting factor. So far, the radiation damage has manifested itself in the form of a loss of overall diffracted intensity and an increase in the temperature factor. The structure of a protein (myrosinase) after exposure to different doses of X-rays in the region of 20 x 10(15) photons mm(-2) has been studied. The changes in the structure owing to radiation damage were analysed using Fourier difference maps and occupancy refinement for the first time. Damage was obvious in the form of breakage of disulfide bonds, decarboxylation of aspartate and glutamate residues, a loss of hydroxyl groups from tyrosine and of the methylthio group of methionine. The susceptibility to radiation damage of individual groups of the same kind varies within the protein. The quality of the model resulting from structure determination might be compromised owing to the presence of radiolysis in the crystal after an excessive radiation dose. Radiation-induced structural changes may interfere with the interpretation of ligand-binding studies or MAD data. The experiments reported here suggest that there is an intrinsic limit to the amount of data which can be extracted from a sample of a given size.

Photochemical inactivation of enzymes.

The mechanisms of enzyme inactivation by ultraviolet light and visible light in the presence of sensitizing dyes are reviewed. Recent flash photolysis studies on amino acids and enzymes are summarized in terms of proposed models relating the initial photochemical reactions to permanent chemical and biological damage. The generation and reactions of singlet oxygen are discussed in connection with photodynamic processes. The photochemical results are compared with ionizing radiations, particularly pulse radiolytic methods employing radical anions as selective probes. The interrelationships between the various modes of enzyme inactivation are discussed, as well as the new information to be learned about the structure and functions of the native enzymes from selective radiation-induced alterations.

Nuclear coupling of 33S and the nature of free radicals in irradiated crystals of cystine dihydrochloride.

The (33)S hyperfine structure has been observed and analyzed in the electron spin resonance of gamma-irradiated single crystals of cystine dihydrochloride at 77 degrees K and at room temperature. The radicals observed at room temperature and previously assigned as RCH(2)S are proved to be disulfide radicals, RCH(2)SS. However, the RCH(2)S radical is identified by the (33)S measurements in samples irradiated at 77 degrees K, annealed at 200 degrees K, and observed at 77 degrees K. This monosulfide radical appears to undergo reactions to form disulfide radicals at room temperature.