Application of fluorescence resonance energy transfer to the GroEL-GroES chaperonin reaction.
Application of fluorescence resonance energy transfer to the GroEL-GroES chaperonin reaction.
复制标题
荧光共振能量转移在 GroEL-GroES 伴侣蛋白反应中的应用。
DOI：
10.1006/meth.2001.1188
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Rye,HS
中科院分区：
文献类型：
--
作者：
Rye,HS
荧光共振能量转移（FRET）是一种灵敏、灵活的研究蛋白质相互作用的方法。在这里，它适用于GroEL-GroES伴侣蛋白系统检查ATP驱动的动力学，该伴侣蛋白折叠的基础。依赖于GroEL和GroES的已知结构，将用于附着荧光探针的位点设计到两种蛋白质的序列中。因为当GroEL和GroES形成复合物时，这些位点在空间上接近，所以激发能量可以通过FRET从供体传递到受体发色团。虽然在理想情况下FRET可以用于测量距离，但GroEL-GroES复合物中供体-受体距离的显著群体异质性使得距离测定困难。这是由于这些大的寡聚蛋白质的不完全标记和它们的旋转对称性。然而，它示出，FRET仍然可以被用来遵循蛋白质-蛋白质相互作用的动力学，即使在这样的情况下，距离测量是不实际的或没有意义的。以这种方式，FRET信号被用作简单的接近传感器来对GroEL和GroES之间的相互作用进行评分。类似地，FRET也可用于跟踪GroEL与荧光标记的底物多肽之间的相互作用。因此，虽然分子结构的知识极大地帮助FRET实验的设计，结构信息是不一定需要的，如果目的是测量的热力学或动力学的蛋白质相互作用的事件，通过以下两个组件的结合接近的变化。
Fluorescence resonance energy transfer (FRET) is a sensitive and flexible method for studying protein-protein interactions. Here it is applied to the GroEL-GroES chaperonin system to examine the ATP-driven dynamics that underlie protein folding by this chaperone. Relying on the known structures of GroEL and GroES, sites for attachment of fluorescent probes are designed into the sequence of both proteins. Because these sites are brought close in space when GroEL and GroES form a complex, excitation energy can pass from a donor to an acceptor chromophore by FRET. While in ideal circumstances FRET can be used to measure distances, significant population heterogeneity in the donor-to-acceptor distances in the GroEL-GroES complex makes distance determination difficult. This is due to incomplete labeling of these large, oligomeric proteins and to their rotational symmetry. It is shown, however, that FRET can still be used to follow protein-protein interaction dynamics even in a case such as this, where distance measurements are either not practical or not meaningful. In this way, the FRET signal is used as a simple proximity sensor to score the interaction between GroEL and GroES. Similarly, FRET can also be used to follow interactions between GroEL and a fluorescently labeled substrate polypeptide. Thus, while knowledge of molecular structure aids enormously in the design of FRET experiments, structural information is not necessarily required if the aim is to measure the thermodynamics or kinetics of a protein interaction event by following changes in the binding proximity of two components.