Bowman Sarah E J, Bridwell-Rabb Jennifer, Drennan Catherine L
Department of Chemistry, ‡Department of Biology, and §Howard Hughes Medical Institute, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
Acc Chem Res. 2016 Apr 19;49(4):695-702. doi: 10.1021/acs.accounts.5b00538. Epub 2016 Mar 15.
Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV-vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.
金属离子和金属辅因子在广泛的生化反应中发挥着重要作用。因此，据估计，多达25%至50%的蛋白质组利用过渡金属离子来执行各种基本功能。金属蛋白中所含的金属离子基于化学性质发挥功能作用，其多样性源于过渡金属可以采用不同的氧化还原状态和几何构型，这由金属的特性和蛋白质环境决定。金属离子与金属辅因子（如血红素和钴胺素）中的有机框架的结合，进一步扩展了金属在生物学中的化学功能。蛋白质支架内金属离子和复杂金属辅因子的三维可视化通常是酶学研究的起点，突出了金属蛋白结构表征的重要性。然而，金属蛋白晶体学面临一些潜在挑战，包括正确引入相关金属或金属辅因子、为蛋白质的纯化和结晶维持合适的环境（包括提供厌氧、低温或无光环境），以及注意X射线对蛋白质或所含金属离子造成损伤的可能性。尽管如此，所含的金属或金属辅因子在金属蛋白晶体学中也具有独特优势。在用于蛋白质晶体学的波长下，金属与X射线光子发生显著共振，且金属具有丰富的电子特性，能提供强烈且具有光谱独特性的信号，这使得金属蛋白晶体学家能够利用反常色散来确定结构解析的相位，并对单晶使用同步或平行光谱技术。这些特性，再加上光束线亮度的提高、调节X射线束波长的能力、先进探测器的可用性以及许多同步加速器光束线配备的光谱设备，在金属蛋白结构测定方面取得了令人兴奋的进展。在此，我们将展示金属在金属蛋白晶体学中的有利应用成果，包括利用金属辅因子获取相位信息、利用K边X射线吸收光谱鉴定金属蛋白晶体中配位的金属，以及对晶体使用紫外可见光谱来探测结晶蛋白的酶活性。
