<br>ReAsH 作为细胞内蛋白质动力学的定量探针,Biochemistry

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ReAsH 作为细胞内蛋白质动力学的定量探针
Biochemistry ( IF 2.9 ) Pub Date : 2016-03-18 00:00:00 , DOI: 10.1021/acs.biochem.5b01336
Hannah Gelman ₁ , Anna Jean Wirth ₁ , Martin Gruebele ₁

Affiliation

四半胱氨酸 (tc) 标签/双砷染料系统（FlAsH 或 ReAsH）有望将荧光蛋白标签的灵活性与小尺寸染料标签相结合，从而允许对受大蛋白标签干扰的目标蛋白进行细胞内研究。使用 FlAsH 和 ReAsH 进行的细胞内定量热力学和动力学研究受到方法学复杂性的阻碍，该复杂性是通过福斯特共振能量转移 (FRET) 或直接激发探测的标签-染料复合物的荧光特性所呈现的。我们用 AcGFP1 和 ReAsH 标记模型蛋白磷酸甘油酸激酶 (PGK)，以便与 AcGFP1/mCherry 标记的 PGK 直接比较。我们发现快速弛豫成像 (FReI) 将毫秒温度跳跃动力学与荧光显微镜检测相结合，避免了使用 ReAsH 系统时遇到的许多困难，使我们能够在体外和细胞中获得蛋白质稳定性和动力学的定量 FRET 测量。我们还向我们展示了令人惊讶的结果，即来自模型蛋白 C 末端直接激发的未埋藏 ReAsH 的荧光也报告了体外和细胞内的折叠。通过将 ReAsH 标记的蛋白质与用两个荧光蛋白质标签标记的构建体进行比较，我们可以评估体积较大的蛋白质标签如何影响细胞内和体外的蛋白质动态。我们发现，细胞中的平均折叠率更接近标签较小的体外折叠率，突出了标签对定量细胞内测量的影响。

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ReAsH as a Quantitative Probe of In-Cell Protein Dynamics

The tetracysteine (tc) tag/biarsenical dye system (FlAsH or ReAsH) promises to combine the flexibility of fluorescent protein tags with the small size of dye labels, allowing in-cell study of target proteins that are perturbed by large protein tags. Quantitative thermodynamic and kinetic studies in-cell using FlAsH and ReAsH have been hampered by methodological complexities presented by the fluorescence properties of the tag–dye complex probed by either Förster resonance energy transfer (FRET) or direct excitation. We label the model protein phosphoglycerate kinase (PGK) with AcGFP1 and ReAsH for direct comparison with AcGFP1/mCherry-labeled PGK. We find that fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells. We also demonstrate the to us surprising result that fluorescence from directly excited, unburied ReAsH at the C-terminus of the model protein also reports on folding in vitro and in cells. Comparing the ReAsH-labeled protein to a construct labeled with two fluorescent protein tags allows us to evaluate how a bulkier protein tag affects protein dynamics in cells and in vitro. We find that the average folding rate in the cell is closer to the in vitro rate with the smaller tag, highlighting the effect of tags on quantitative in-cell measurements.

更新日期：2016-03-18

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