Wong Katy A, O'Bryan John P
Department of Pharmacology, University of Illinois at Chicago, USA.
J Vis Exp. 2011 Apr 15(50):2643. doi: 10.3791/2643.
Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1). A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET). For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.
确定信号复合物的亚细胞分布对于理解该复合物的输出至关重要。免疫沉淀等传统方法无法提供复合物空间定位的信息。相比之下，双分子荧光互补（BiFC）可监测蛋白质复合物的相互作用和亚细胞区室化。在这种方法中，荧光蛋白被拆分为氨基末端和羧基末端的非荧光片段，然后与两个感兴趣的蛋白质融合。蛋白质的相互作用导致荧光团的重构（图1）。BiFC的一个局限性在于，一旦片段化的荧光团重构，复合物就不可逆转。这一局限性在检测瞬时或弱相互作用时具有优势，但排除了对复合物动力学的动力学分析。另一个需要注意的问题是，重构的荧光团需要30分钟才能成熟并发出荧光，这同样排除了对实时相互作用的观察。BiFC是蛋白质片段互补分析（PCA）的一个具体例子，PCA采用绿色荧光蛋白变体（BiFC）、二氢叶酸还原酶、β-内酰胺酶和荧光素酶等报告蛋白来测量蛋白质与蛋白质之间的相互作用。研究细胞中蛋白质与蛋白质相互作用的其他方法包括荧光共定位和荧光共振能量转移（FRET）。对于共定位，两种蛋白质分别直接用荧光团标记或通过间接免疫荧光标记。然而，这种方法会导致非相互作用蛋白质的高背景，使得难以解释共定位数据。此外，由于共聚焦显微镜分辨率的限制，两种蛋白质可能看起来共定位但不一定相互作用。使用BiFC时，只有当两个感兴趣的蛋白质相互作用时才会观察到荧光。FRET是研究蛋白质与蛋白质相互作用的另一种出色方法，但在技术上可能具有挑战性。FRET实验要求供体和受体在细胞中具有相似的亮度和化学计量比。此外，必须考虑供体渗漏到受体通道以及反之亦然的情况。与FRET不同，BiFC背景荧光很少，图像数据后处理很少，不需要高表达，并且可以检测弱或瞬时相互作用。生物发光共振能量转移（BRET）是一种类似于FRET的方法，不同之处在于供体是一种酶（如荧光素酶），它催化底物发出生物发光从而激发受体。BRET没有渗漏和高背景荧光的技术问题，但由于缺乏底物定位于特定区室的能力，无法提供空间信息。总体而言，BiFC是一种用于可视化蛋白质复合物亚细胞定位以深入了解区室化信号传导的出色方法。
