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，这种假体需要跨细胞膜运输，这表明存在高度控制的递送机制。此外，关于血红素信号传导和调控特性的越来越多的证据表明，这种无处不在的辅助因子的作用范围比传统认为的要广泛得多。为了阐明血红素在细胞代谢和体内平衡中的作用，需要对不稳定血红素库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建造一个传感器，用于定量测定细胞中不稳定血红素的浓度。这将涉及荧光标记蛋白的构建，该蛋白可以在一系列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，试图追踪血红素进入细胞。重组蛋白将主要在大肠杆菌中表达，利用适当的工程质粒载体。纯化和鉴定将涉及标准的分子生物学程序。血红素传感器结合和成像研究的定量将需要使用光谱/成像技术，如FLIM和FLIM- fret。该项目由布里斯托尔大学的Emma Raven教授监督，并将涉及Andrew Hudson博士（莱斯特大学）和Wolfson生物医学中心（布里斯托尔）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).