Grzyb Joanna, Xu Fei, Weiner Lev, Reijerse Eduard J, Lubitz Wolfgang, Nanda Vikas, Noy Dror
Plant Sciences Department, Weizmann Institute of Science, Rehovot, Israel.
Biochim Biophys Acta. 2010 Mar;1797(3):406-13. doi: 10.1016/j.bbabio.2009.12.012. Epub 2009 Dec 24.
Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe(4)S(4)) cluster protein with a non-natural alpha-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe(4)S(4) cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe(4)S(4) clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, alpha-helical coiled-coil binding a single Fe(4)S(4) cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, Fe(4)S(4) (S=1/2), with g values of g(zy)=(1.970, 1.975), and g(x)=2.053. The temperature, and power dependence of the signal intensity were also characteristic of Fe(4)S(4) clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes.
我们采用“金属优先”方法,通过计算设计、制备并表征了一种具有非天然α-螺旋卷曲螺旋结构的四铁四硫(Fe(4)S(4))簇蛋白。这种结构的新颖之处在于Fe(4)S(4)簇位于四螺旋束的疏水核心内,这使其在先前的铁硫(FeS)蛋白设计中独一无二,且不同于已知的天然FeS蛋白。该脱辅基蛋白从大肠杆菌中重组表达并纯化,在体外能轻易地与Fe(4)S(4)簇自组装。紫外可见吸收光谱、圆二色光谱、元素分析、凝胶过滤和分析超速离心证实该蛋白按设计折叠并组装,即α-螺旋卷曲螺旋结合单个Fe(4)S(4)簇。连二亚硫酸盐还原的全蛋白样品具有典型的菱形电子顺磁共振光谱,属于低电位的Fe(4)S(4)(S = 1/2),g值为g(zy)=(1.970, 1.975),g(x)=2.053。信号强度的温度和功率依赖性也具有Fe(4)S(4)簇的特征,自旋弛豫非常有效,但相邻簇之间几乎没有任何相互作用。尽管需要优化,特别是防止聚集以及添加二级壳层相互作用以稳定还原态,但这种新设计很有前景。其主要优点是通过简单地沿卷曲螺旋轴复制和翻译结合位点,可扩展为多FeS簇蛋白。这为设计可作为“导线”用于连接任何给定的一组氧化还原酶的蛋白嵌入氧化还原链开辟了新的可能性。