FRET-capture: a sensitive method for the detection of dynamic protein interactions.
FRET-capture: a sensitive method for the detection of dynamic protein interactions.
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DOI：
10.1002/cbic.201200700
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发表时间：
2013-01-02
期刊：
影响因子：
3.2
通讯作者：
Imperiali, Barbara
中科院分区：
文献类型：
--
作者：
Socher, Elke;Imperiali, Barbara
识别和分析大分子过程，如蛋白质-蛋白质相互作用和构象动力学是化学生物学、生物化学和医学的核心。基于荧光的传感器是监测这些过程的重要工具。大多数这样的传感器依赖于FRET，它利用两个荧光团的距离依赖的相互作用来报告接近和/或荧光团偶极相互作用的变化。FRET已被广泛用于探索各种生化现象的时空调节基于fret系统的设计涉及两个荧光团的战略定位，以最大限度地提高信号变化，使研究人员能够监测生物事件。理想情况下，基于FRET的测定导致供体发射的减少和受体发射的增加，允许FRET过程之后的比例测量。荧光标记既可以融合到两个相互作用的伙伴以报告分子组装（分子间FRET），也可以标记在同一蛋白质上以报告构象变化（分子内FRET）。将荧光探针整合到蛋白质中是很普遍的，通常使用基因编码的自荧光蛋白（FPs）对，如GFP变体CFP和YFP，分别作为供体和受体荧光团一般来说，使用分子内FRET的可接受的发射强度变化只能通过不同结构的大量实验来实现，并且通常，由于高背景荧光，信号与背景比很小。此外，FPs的大小（~ 27 kDa）可能会干扰蛋白质定位并使FRET分析复杂化另外，通过自标记蛋白质标签（如SNAP-tag、[4]CLIP-tag、[5]或Halo-tag[6]）将合成荧光团附着在目标蛋白质上的方法已被证明是有价值的。在这种情况下，可以招募的合成荧光团的范围相当大。然而，由于存在未反应探针和非特异性结合，这些系统的一个限制是高荧光背景信号。因此，清洗步骤可能是必要的；然而，这些过程并不总是可能的，例如在绑定事件的实时度量中。在这种情况下，最近报道了一些减少背景荧光的有用方法一般来说，基于fret的方法需要相当大的分子间或分子内距离调制来观察荧光的有用变化。虽然在某些情况下，可以设计目标系统来增强光谱变化，例如最近开发的基于碳酸酐酶的FRET系统用于传感苯磺酰胺，但仍然有机会采用新的实验方法来测量动态蛋白质相互作用和构象变化。在此，我们提出了一种替代FRET方法的设计、开发和验证，该方法利用溶剂致变色荧光团作为FRET供体。这种方法解决了传统的基于fret的实验的一些缺点。在这种情况下，我们注意到研究DNA序列特异性识别的方法，利用嵌入的环境敏感荧光团作为FRET供体已被报道溶剂致变色荧光团，如二甲氨基萘酰亚胺（DMNs）和二甲氨基酞酰亚胺（DMPs）对局部环境敏感，在极性质子环境中表现出极弱的荧光，从而具有低背景信号的优势，直到发生改变局部环境的事件。
Identifying and analyzing macromolecular processes such as protein–protein interactions and conformational dynamics are central to chemical biology, biochemistry and medicine. Fluorescence-based sensors are important tools for monitoring these processes. The majority of such sensors rely on FRET, which takes advantage of the distance-dependent interaction of two fluorophores to report proximity and/or changes in the fluorophore dipolar interactions. FRET has been extensively used to explore spatiotemporal regulation of various biochemical phenomena.[1] The design of FRET-based systems involves strategic positioning of two fluorophores to maximize signal changes enabling the researcher to monitor biological events. Ideally, the FRET-based assays lead to a decrease in the donor emission and an increase of the acceptor emission, allowing FRET processes to be followed by ratiometric measurements. Fluorescent labels can either be fused to two interacting partners to report molecular assembly (intermolecular FRET) or tagged on the same protein to report conformational changes (intramolecular FRET). The integration of fluorescent probes into proteins is widespread and often pairs of genetically encoded autofluorescent proteins (FPs) such as the GFP variants CFP and YFP, as donor and acceptor fluorophores, respectively, are applied.[2] In general, acceptable changes in emission intensities using intramolecular FRET can only be achieved by considerable experimentation with different constructs and often, the signal to background ratios are small due to high background fluorescence. Also, the size (∼ 27 kDa) of the FPs may perturb protein localization and complicate FRET analyses.[3] Alternatively, approaches where synthetic fluorophores are attached to target proteins through self-labeling protein tags such as the SNAP-tag,[4] CLIP-tag,[5] or Halo-tag [6] have proven to be valuable. In this case, the range of synthetic fluorophores that can be recruited is considerable. However, one limitation of these systems is high fluorescence background signal due to the presence of unreacted probes and nonspecific binding. Therefore washing steps may be necessary; however, these processes are not always possible, for example in the real-time measurement of binding events. In this context, useful approaches for reducing background fluorescence have recently been reported.[7] In general, FRET-based approaches necessitate considerable inter-or intramolecular distance modulation to observe useful changes in fluorescence. While in some cases the target systems can be engineered to enhance spectral changes, for example in the recent development of a carbonic anhydrase-based FRET system for sensing benzenesulfonamide,[8] there remains the opportunity for new experimental approaches for measuring dynamic protein interactions and conformational changes.Herein, we present the design, development and validation of an alternative FRET approach that exploits a solvatochromic fluorophore as a FRET donor. This approach addresses some of the shortcomings of traditional FRET-based experiments. In this context we note that methods for investigating the sequencespecific recognition of DNA, by exploiting intercalated environment-sensitive fluorophores as FRET donors have been reported.[9] Solvatochromic fluorophores such as dimethylaminonaphthalimides (DMNs) and dimethylaminophthalimides (DMPs) are sensitive to the local environment and exhibit extremely weak fluorescence in polar protic environments conferring the advantage of low background signals until the occurrence of an event that alters the local …
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