Diabetes Center, University of California, San Francisco, California 94143-0540, USA.
J Biol Chem. 2010 Jan 22;285(4):2428-37. doi: 10.1074/jbc.M109.045203. Epub 2009 Nov 19.
An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor alpha-isoform (ER alpha) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER alpha with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER alphas at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ER alpha monomer at higher SRC-RID concentrations (approximately 10 nm). The methods also showed that only a subpopulation of ER alpha was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment.
能够在细胞环境中的特定位置测量蛋白质复合物的生化参数和结构，将有助于我们深入理解细胞功能。我们描述了广泛适用的、经过校准的Förster 共振能量转移方法，这些方法可定量测量人类雌激素受体α-异构体（ERα）与三种共因子（SRC1、SRC2、SRC3）的受体相互作用域（RID）在活细胞中的结构和生化参数。在整个细胞核中测量到的 ERα 与所有三种 SRC-RID 的相互作用，从在低游离 SRC-RID 浓度（<2nm）下含有两个 ERα 的结构相似、高亲和力复合物，转变为在较高 SRC-RID 浓度（约 10nm）下具有 ERα 单体的低亲和力复合物。这些方法还表明，只有一小部分 ERα 可与细胞中的 SRC-RID 形成复合物。这些方法为提取任何能够在细胞环境中通过 Förster 共振能量转移进行测量的复合物的生化和结构的前所未有的细节提供了模板。
