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Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins
Biomolecules ( IF 5.5 ) Pub Date : 2020-10-19 , DOI: 10.3390/biom10101458 Anna Morató 1 , Carlos A Elena-Real 1 , Matija Popovic 1 , Aurélie Fournet 1 , Karen Zhang 1 , Frédéric Allemand 1 , Nathalie Sibille 1 , Annika Urbanek 1 , Pau Bernadó 1
Biomolecules ( IF 5.5 ) Pub Date : 2020-10-19 , DOI: 10.3390/biom10101458 Anna Morató 1 , Carlos A Elena-Real 1 , Matija Popovic 1 , Aurélie Fournet 1 , Karen Zhang 1 , Frédéric Allemand 1 , Nathalie Sibille 1 , Annika Urbanek 1 , Pau Bernadó 1
Affiliation
The high-resolution structural study of huntingtin exon-1 (HttEx1) has long been hampered by its intrinsic properties. In addition to being prone to aggregate, HttEx1 contains low-complexity regions (LCRs) and is intrinsically disordered, ruling out several standard structural biology approaches. Here, we use a cell-free (CF) protein expression system to robustly and rapidly synthesize (sub-) pathological HttEx1. The open nature of the CF reaction allows the application of different isotopic labeling schemes, making HttEx1 amenable for nuclear magnetic resonance studies. While uniform and selective labeling facilitate the sequential assignment of HttEx1, combining CF expression with nonsense suppression allows the site-specific incorporation of a single labeled residue, making possible the detailed investigation of the LCRs. To optimize CF suppression yields, we analyze the expression and suppression kinetics, revealing that high concentrations of loaded suppressor tRNA have a negative impact on the final reaction yield. The optimized CF protein expression and suppression system is very versatile and well suited to produce challenging proteins with LCRs in order to enable the characterization of their structure and dynamics.
中文翻译:
用于NMR研究的亚病理和病理性Huntingtin Exon-1的可靠的无细胞表达。低复杂度蛋白质同位素标记的一般方法
亨廷顿外显子1(HttEx1)的高分辨率结构研究长期以来一直受其固有特性的阻碍。除了易于聚集外,HttEx1还包含低复杂度区域(LCR),并且本质上无序,从而排除了几种标准的结构生物学方法。在这里,我们使用无细胞(CF)蛋白质表达系统来稳健,快速地合成(亚)病理学HttEx1。CF反应的开放性允许应用不同的同位素标记方案,从而使HttEx1适合进行核磁共振研究。虽然统一和选择性标记有助于HttEx1的顺序分配,但将CF表达与无意义的抑制相结合可实现单个标记残基的位点特异性掺入,从而可能对LCR进行详细研究。为了优化CF抑制产量,我们分析了表达和抑制动力学,揭示了高浓度的抑制tRNA对最终反应的产量有负面影响。优化的CF蛋白表达和抑制系统用途广泛,非常适合使用LCR生产具有挑战性的蛋白,从而能够表征其结构和动力学。
更新日期:2020-10-19
中文翻译:
用于NMR研究的亚病理和病理性Huntingtin Exon-1的可靠的无细胞表达。低复杂度蛋白质同位素标记的一般方法
亨廷顿外显子1(HttEx1)的高分辨率结构研究长期以来一直受其固有特性的阻碍。除了易于聚集外,HttEx1还包含低复杂度区域(LCR),并且本质上无序,从而排除了几种标准的结构生物学方法。在这里,我们使用无细胞(CF)蛋白质表达系统来稳健,快速地合成(亚)病理学HttEx1。CF反应的开放性允许应用不同的同位素标记方案,从而使HttEx1适合进行核磁共振研究。虽然统一和选择性标记有助于HttEx1的顺序分配,但将CF表达与无意义的抑制相结合可实现单个标记残基的位点特异性掺入,从而可能对LCR进行详细研究。为了优化CF抑制产量,我们分析了表达和抑制动力学,揭示了高浓度的抑制tRNA对最终反应的产量有负面影响。优化的CF蛋白表达和抑制系统用途广泛,非常适合使用LCR生产具有挑战性的蛋白,从而能够表征其结构和动力学。