Protein Biosensors with Customized Properties for Live-Cell Imaging

描述（由申请人提供）：活细胞荧光显微镜提供了细胞中发生的动态过程的空间分辨动力学信息，因此它在信号转导和其他细胞内过程的定量测量方面具有独特的潜力。然而，这种方法还没有充分发挥其潜力，因为它目前受到充分表征的生物传感器分子的可用性的限制，这些分子可以通过基因编码或微注射到细胞中，并且可以识别感兴趣的细胞内靶分子。大多数活细胞成像从业者没有意识到的是，一个合适的生物传感器对其靶标的结合亲和力必须在一个相对狭窄的范围内，并且依赖于来自天然存在的蛋白质的模块化结合域在这方面提出了一个主要的限制。在此，我们建议合作开发一种新的通用方法，使具有活细胞成像规定特性的细胞内生物传感器能够重新设计。要做到这一点，很明显，我们必须摆脱来自自然产生的蛋白质的结合域。相反，我们的出发点是一个由7种不同的蛋白质组成的集合，这些蛋白质来自嗜热古菌和细菌（它们在极端温度下茁壮成长），作为工程生物传感器的模板或支架。这些支架有几个有利的特性；它们分子量低（约100个氨基酸或更少），缺乏二硫键，在哺乳动物细胞中功能惰性。具有所需结合特异性的蛋白质将从我们通过蛋白质支架表面可接近残基的随机化成功生成的大量（bbb108）突变蛋白组合集合中筛选。由于蛋白质工程策略使用多个支架，我们将变体库称为超级库，它具有比a更大的理论多样性

DESCRIPTION (provided by applicant): Live-cell fluorescence microscopy provides spatially resolved kinetic information about dynamic processes as they occur in cells, and therefore it has unique potential for the quantitative measurement of signal transduction and other intracellular processes. This approach has not yet lived up to its full potential, however, because it is currently limited by the availability of well-characterized biosensor molecules that can be either genetically encoded or microinjected into cells and which recognize intracellular target molecules of interest. What is not appreciated by most practitioners of live-cell imaging is that the binding affinity of a suitable biosensor for its target(s) must lie within a relatively narrow range, and the reliance on modular binding domains derived from naturally occurring proteins presents a major constraint in that regard. Herein, we propose a collaborative effort to develop a novel and general approach enabling de novo design of intracellular biosensors with prescribed properties for live-cell imaging. To do this, it is clear that we must move away from binding domains derived from naturally occurring proteins. Instead, our starting point is an ensemble of 7 different proteins from hyperthermophilic archaea and bacteria (which thrive at extreme temperatures) as templates, or scaffolds, for engineering biosensors. These scaffolds have several favorable properties; they are low in molecular weight (~100 amino acids or less), lack disulfide bonds, and are functionally inert in mammalian cells. Proteins with desired binding specificity will be screened from a large (> 108) combinatorial collection of mutant proteins that we have successfully generated through randomization of surface-accessible residues on the protein scaffolds. Because the protein engineering strategy uses multiple scaffolds, we refer to the repertoire of variants as a super library, which possesses greater theoretical diversity than a library of the same size derived from any single scaffold. We propose to screen the super library for biosensors recognizing specific phosphorylation sites and characterize their binding affinities (Aim 2). Subsequently, we will validate the identified biosensors by characterizing thei translocation in cells expressing wild-type or mutant Epidermal Growth Factor Receptor (EGFR) (Aim 2). Finally, we propose a pilot study to assess the kinetics of site-specific EGFR phosphorylation in human mammary epithelial cells (Aim 3).