TR-FRET Assays of Huntingtin Protein Fragments Reveal Temperature and PolyQ Length-Dependent Conformational Changes

被引：15

作者：

Cui, Xiaotian ^{[1
]}

Liang, Qingnan ^{[1
]}

Liang, Yijian ^{[1
]}

Lu, Mingxing ^{[1
]}

Ding, Yu ^{[1
]}

Lu, Boxun ^{[1
]}

机构：

[1] Fudan Univ, Sch Life Sci, State Key Lab Genet Engn, Shanghai 200433, Peoples R China

来源：

SCIENTIFIC REPORTS | 2014年 / 4卷

关键词：

MUTANT HUNTINGTIN; MONOCLONAL-ANTIBODIES; POLYGLUTAMINE; EXON-1; BINDING; MODEL;

D O I：

10.1038/srep05601

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

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 degrees 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.

引用

页数：8