Protein sensor design for the quantification of heme concentration in cells

由于血红素金属卟啉的潜在毒性和疏水性，该辅基跨细胞膜运输的必要性表明存在高度受控的递送机制。此外，积累的证据的信号和调节特性的血红素是一个更广泛的范围内的作用，这种无处不在的辅因子比传统上认为。为了阐明血红素在细胞代谢和稳态中的作用，需要对不稳定血红素池的生物学意义有新的认识。作为对这一宏伟计划的贡献，该项目的目标是设计和构建一种用于量化细胞中不稳定血红素浓度的传感器。这将涉及构建荧光标记的蛋白质，其可以在一定范围的血红素浓度和条件下结合血红素。此外，融合血红素结合蛋白的光学特性将被用于成像目的，试图跟踪血红素进入细胞。大肠杆菌利用适当改造的质粒载体。纯化和表征将涉及标准分子生物学程序。血红素传感器结合的定量和成像研究将需要使用光谱/成像技术，如FLIM和FLIM-FRET。该项目是在布里斯托大学的Emma Raven教授的监督下进行的，并将涉及Andrew哈德逊博士（莱斯特大学）和Wolfson生物老化中心（布里斯托）的合作。

Due to the potentially toxic nature and hydrophobicity of the heme metalloporphyrin, the necessity for this prosthetic group to be trafficked across cell membranes suggests the existence of highly controlled delivery mechanisms. Furthermore, accumulating evidence on the signaling and regulatory properties of heme is indicative of a much wider spectrum of roles for this ubiquitous cofactor than traditionally thought. New insights into the biological significance of the labile heme pool are required in order to elucidate heme roles within the cell metabolism and homeostasis. As a contribution to this grand scheme, the aim of the project is to design and build a sensor for the quantification of labile heme concentration in cells. This will involve the construction of fluorescently-labelled proteins which can bind heme under a range of heme concentrations and conditions. Moreover, the optical properties of the fusion heme-binding proteins will be exploited for imaging purposes, trying to track heme into cells.Recombinant proteins will be mainly expressed in E. Coli making use of properly engineered plasmid vectors. Purification and characterization will involve standard molecular biology procedures. The quantification of the heme-sensor binding and the imaging studies will require the use spectroscopy/imaging techniques such as FLIM and FLIM-FRET.The project is under the supervision of Prof. Emma Raven at the University of Bristol and will involve the collaboration of Dr. Andrew Hudson (Leicester University) and the Wolfson Biomaging center (Bristol).