TR-FRET assays of Huntingtin protein fragments reveal temperature and polyQ length-dependent conformational changes.
TR-FRET assays of Huntingtin protein fragments reveal temperature and polyQ length-dependent conformational changes.
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亨廷顿蛋白片段的 TR-FRET 分析揭示了温度和 PolyQ 长度依赖性构象变化
DOI：
10.1038/srep05601
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发表时间：
2014-07-07
影响因子：
4.6
通讯作者：
Lu B
中科院分区：
文献类型：
--
作者：
Cui X;Liang Q;Liang Y;Lu M;Ding Y;Lu B
时间分辨荧光共振能量转移（TR-FRET）技术是一种广泛使用的免疫测定技术，能够高通量定量测量感兴趣的蛋白质。其中一个已建立的例子是突变亨廷顿蛋白（HTT）的TR-FRET测定，其是神经退行性亨廷顿病（HD）的主要原因。为了测量突变体HTT蛋白，已发表的测定法利用与HTT N-末端抗体配对的polyQ抗体MW1。MW1对具有扩展polyQ链段的突变HTT的表观亲和力比对具有较短polyQ的野生型HTT的表观亲和力高得多，因此该测定优先检测突变HTT。在这里，我们报告了HTT N-末端片段的TR-FRET信号的可逆温度依赖性变化：当温度从室温降低到4 ° C时，信号变得更高。有趣的是，TR-FRET信号的温度敏感性对于Q25（野生型）比对于Q72（突变型）蛋白高得多。我们进一步揭示，这可能是由于HTT的温度和polyQ长度依赖性结构或空间变化，这对于理解polyQ结构和毒性可能是有用的。
Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) technology is a widely used immunoassay that enables high-throughput quantitative measurements of proteins of interest. One of the well established examples is the TR-FRET assay for mutant huntingtin protein (HTT), which is the major cause of the neurodegenerative Huntington's disease (HD). To measure the mutant HTT protein, the published assays utilize a polyQ antibody, MW1, paired with HTT N-terminal antibodies. MW1 has much higher apparent affinity to mutant HTT with expanded polyQ stretch than to wild-type HTT with shorter polyQ and thus the assays detect mutant HTT preferentially. Here we report a reversible temperature dependent change of TR-FRET signals for HTT N-terminal fragments: the signals become higher when the temperature is lowered from room temperature to 4°C. Interestingly, the temperature sensitivity of the TR-FRET signals is much higher for the Q25 (wild-type) than for the Q72 (mutant) protein. We further revealed that it is likely due to a temperature and polyQ length-dependent structural or spatial change of HTT, which is potentially useful for understanding polyQ structure and toxicity.
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DOI：
10.1073/pnas.1221891110
发表时间：
2013-02-05
影响因子：
11.1
作者：
Sathasivam, Kirupa;Neueder, Andreas;Bates, Gillian P.
通讯作者：
Bates, Gillian P.
影响因子：
4.8
作者：
Baldo, Barbara;Weiss, Andreas;Kaupmann, Klemens
通讯作者：
Kaupmann, Klemens
影响因子：
2.9
作者：
CHANG, CT;WU, CSC;YANG, JT
通讯作者：
YANG, JT
影响因子：
16.8
作者：
Li, Pingwei;Huey-Tubman, Kathryn E.;Bjorkman, Pamela J.
通讯作者：
Bjorkman, Pamela J.
影响因子：
25
作者：
Lu, Boxun;Al-Ramahi, Ismael;Palacino, James
通讯作者：
Palacino, James