The susceptibility of disulfide bonds towards radiation damage may be explained by S⋯O interactions

Abstract

Radiation-induced damage to protein crystals during X-ray diffraction data collection is a major impediment to obtaining accurate structural information on macromolecules. Some of the specific impairments that are inflicted upon highly brilliant X-ray irradiation are metal-ion reduction, disulfide-bond cleavage and a loss of the integrity of the carboxyl groups of acidic residues. With respect to disulfide-bond reduction, previous results have indicated that not all disulfide bridges are equally susceptible to damage. A careful analysis of the chemical environment of disulfide bonds in the structures of elastase, lysozyme, acetylcholinesterase and other proteins suggests that S-S bonds which engage in a close contact with a carbonyl O atom along the extension of the S-S bond vector are more susceptible to reduction than the others. Such an arrangement predisposes electron transfer to occur from the O atom to the disulfide bond, leading to its reduction. The interaction between a nucleophile and an electrophile, akin to hydrogen bonding, stabilizes protein structures, but it also provides a pathway of electron transfer to the S-S bond, leading to its reduction during exposure of the protein crystal to an intense X-ray beam. An otherwise stabilizing interaction can thus be the cause of destabilization under the condition of radiation exposure.

Keywords: NBO; S⋯O interactions; disulfide bonds; electron transfer; quantum-chemical calculations; radiation damage.

Figure 1

Spherical polar angles (θ, φ) defining the position of the carbonyl O atom relative to the disulfide plane. Of the two S atoms, that making the contact is labeled S^γ and the other is labeled S^γ′. The figure is based on the convention used in Bhattacharyya et al. (2004 ▸).

Figure 2

Model of CH₃-S-S-CH₃ interacting with CH₃-CO-NH-CH₃ at θ = 90°, φ = −60° and S⋯O distance = 3.08 Å. The spherical polar angles as defined in Fig. 1 ▸ are indicated relative to the normal to the plane and the bisector to the angle S^γ′—S^γ—C^β.

Figure 3

Ribbon diagram of elastase with a ball-and-stick representation of the disulfide bonds. All of the disulfide-bridged cysteine residues have zero relative solvent accessibilities. The detailed environment of the Cys30–Cys46 disulfide bond is shown in (b).

Figure 4

(a) Scatter plot (in stereo) of the distribution of carbonyl O atoms around the disulfide plane in elastase; the atoms interacting with all four disulfide bonds from the first data set (A-0) are shown. The C^β—S^γ—S^γ′ plane (where S^γ is the atom in contact with O) is the common frame around which the coordinates of the O atoms are expressed; these are labeled with the numbers of the residues that they belong to (given in Table 2 ▸). (b) Polar graph of θ versus φ values taken from Tables 2 ▸, 4 ▸ and 5 ▸: those for the susceptible disulfide bonds are in blue (if there are multiple contacts, the first entry is used), and the less susceptible disulfide bonds are in red (that with the shortest contact distance is used if there are multiple entries).

Figure 5

(a) The preferred directions of interaction of an electrophile and a nucleophile with respect to the disulfide plane. (An electrophile can also interact with the other lone-pair orbital.) (b) The incipient reaction of a nucleophile (the carbonyl group) with the disulfide group. (c) Schematic representation of the orbital interaction [n_O→σ*_{(Sγ—Sγ′)}] revealed by NBO calculations.