Holton James M
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158-2330, USA.
J Synchrotron Radiat. 2007 Jan;14(Pt 1):51-72. doi: 10.1107/S0909049506048898. Epub 2006 Dec 15.
The radiation-induced disordering of selenomethionine (SeMet) side chains represents a significant impediment to protein structure solution. Not only does the increased B-factor of these sites result in a serious drop in phasing power, but some sites decay much faster than others in the same unit cell. These radio-labile SeMet side chains decay faster than high-order diffraction spots with dose, making it difficult to detect this kind of damage by inspection of the diffraction pattern. The selenium X-ray absorbance near-edge spectrum (XANES) from samples containing SeMet was found to change significantly after application of X-ray doses of 10-100 MGy. Most notably, the sharp ;white line' feature near the canonical Se edge disappears. The change was attributed to breakage of the Cgamma-Se bond in SeMet. This spectral change was used as a probe to measure the decay rate of SeMet with X-ray dose in cryo-cooled samples. Two protein crystal types and 15 solutions containing free SeMet amino acid were examined. The damage rate was influenced by the chemical and physical condition of the sample, and the half-decaying dose for the selenium XANES signal ranged from 5 to 43 MGy. These decay rates were 34- to 3.8-fold higher than the rate at which the Se atoms interacted directly with X-ray photons, so the damage mechanism must be a secondary effect. Samples that cooled to a more crystalline state generally decayed faster than samples that cooled to an amorphous solid. The single exception was a protein crystal where a nanocrystalline cryoprotectant had a protective effect. Lowering the pH, especially with ascorbic or nitric acids, had a protective effect, and SeMet lifetime increased monotonically with decreasing sample temperature (down to 93 K). The SeMet lifetime in one protein crystal was the same as that of the free amino acid, and the longest SeMet lifetime measured was found in the other protein crystal type. This protection was found to arise from the folded structure of the protein molecule. A mechanism to explain observed decay rates involving the damaging species following the electric field lines around protein molecules is proposed.
辐射诱导的硒代蛋氨酸(SeMet)侧链无序化是蛋白质结构解析的一个重大障碍。这些位点的B因子增加不仅会导致相位解析能力严重下降,而且在同一晶胞中,一些位点的衰变速度比其他位点快得多。这些对辐射敏感的SeMet侧链随剂量的衰变速度比高阶衍射斑点快,因此很难通过检查衍射图样来检测这种损伤。发现施加10 - 100 MGy的X射线剂量后,含SeMet样品的硒X射线吸收近边光谱(XANES)发生了显著变化。最显著的是,标准Se边附近的尖锐“白线”特征消失了。这种变化归因于SeMet中Cγ - Se键的断裂。这种光谱变化被用作探针来测量低温冷却样品中SeMet随X射线剂量的衰变率。研究了两种蛋白质晶体类型和15种含有游离SeMet氨基酸的溶液。损伤率受样品的化学和物理条件影响,硒XANES信号的半衰期剂量范围为5至43 MGy。这些衰变率比Se原子直接与X射线光子相互作用的速率高34至3.8倍,因此损伤机制必定是一种次级效应。冷却至更结晶状态的样品通常比冷却至非晶态固体的样品衰变更快。唯一的例外是一种蛋白质晶体,其中纳米晶冷冻保护剂具有保护作用。降低pH值,尤其是用抗坏血酸或硝酸,具有保护作用,并且SeMet寿命随样品温度降低(低至93 K)而单调增加。一种蛋白质晶体中SeMet的寿命与游离氨基酸的寿命相同,而测量到的最长SeMet寿命出现在另一种蛋白质晶体类型中。发现这种保护源于蛋白质分子的折叠结构。提出了一种机制来解释观察到的衰变率,该机制涉及沿着蛋白质分子周围电场线的损伤物种。