文章基本信息

标题：TR-FRET Assays of Huntingtin Protein Fragments Reveal Temperature and PolyQ Length-Dependent Conformational Changes
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作者：Xiaotian Cui ; Qingnan Liang ; Yijian Liang 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2014
卷号：4
DOI：10.1038/srep05601
出版社：Springer Nature
摘要：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.