title: TR-FRET assays of Huntingtin protein fragments reveal temperature and polyQ length-dependent conformational changes.
abstract: 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.
authors.affiliation: 1] State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China [2].
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Cui
authors.affiliation: 1] State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China [2].
authors.forename: Qingnan
authors.initials: Q
authors.lastname: Liang
authors.affiliation: 1] State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China [2].
authors.forename: Yijian
authors.initials: Y
authors.lastname: Liang
authors.affiliation: 1] State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China [2].
authors.forename: Mingxing
authors.initials: M
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Ding
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China.
authors.forename: Boxun
authors.initials: B
authors.lastname: Lu
mesh_terms: D005091:Exons; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D000071058:Huntingtin Protein; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012995:Solubility; D044366:Transition Temperature
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010455:Peptides; C097188:polyglutamine