Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Am Chem Soc. 2010 Nov 10;132(44):15516-8. doi: 10.1021/ja107487b.
The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.
尽管人们对驱动膜蛋白折叠和聚集的因素有了新的认识，但从头设计膜蛋白仍然很困难。我们设计了一种膜蛋白 PRIME（卟啉在膜中），它将两个非天然铁二苯基卟啉（Fe(III)DPP）置于足够近的位置，为跨膜电子转移提供了一个多中心途径。先前用于设计多卟啉水溶性螺旋蛋白的计算方法被扩展到这个膜靶标。四个螺旋以 D(2)对称束排列，以双组氨酸几何形状结合两个 Fe(II/III)二苯基卟啉，进一步通过第二壳层氢键稳定。紫外可见吸收光谱、圆二色光谱、分析超速离心、氧化还原电位和 EPR 表明 PRIME 以预期的几何形状高亲和力和特异性结合辅因子。
