Specific chemical and structural damage to proteins produced by synchrotron radiation - PubMed (original) (raw)

Comparative Study

Specific chemical and structural damage to proteins produced by synchrotron radiation

M Weik et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

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Figures

Figure 1

Figure 1

Sequential Fourier maps showing the time course of cleavage of the Cys-254–Cys-265 disulfide bond in _Tc_AChE. Cysteine residues were refined as alanine residues to avoid model bias. (a) 3Fo-2Fc maps, contoured at 1.5 σ. (b) Fo-Fc maps, contoured at 3 σ.

Figure 2

Figure 2

Sequential Fourier maps showing the time course of structural changes in the Cys-402–Cys-521 disulfide bond in _Tc_AChE. Data collection and refinement were as in Fig. 1. (a) 3Fo-2Fc maps, contoured at 1.5 σ. (b) Fo-Fc maps, contoured at 3 σ.

Figure 3

Figure 3

Histogram showing the increase in B factors for the side chains of the different types of amino acid in _Tc_AChE as a consequence of synchrotron irradiation. The horizontal line indicates the mean increase in side-chain B factors. The numbers along the _x_-axis show the number of occurrences of each type of amino acid in _Tc_AChE. The individual bars show the average increase in B factor for each type of amino acid for the second data set (B), as compared with the first data set (A), namely (B factorB − B factorA)/B factorA. Data in this figure, as well as values for increases in B factors mentioned in the text, are derived from models in which the six Sγ atoms of cysteine residues participating in intrachain disulfide linkages were included in the refinement.

Figure 4

Figure 4

Sequential Fourier maps and difference Fourier maps showing cleavage of the Cys-30–Cys-115 disulfide bond in HEWL as a function of x-ray dose at 1.2-Å resolution. (A) Initial 2Fo-Fc map, contoured at 1.5 σ. (B) Difference Fourier map, wFA − wFB, for two successive data sets collected using an attenuator, contoured at 5 σ (the w refers to

sigmaa

weighting (39). (c) Difference Fourier map, wFB − wFC, for two successive data sets, between which the crystal was exposed to the unattenuated beam, contoured at 6 σ.

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