Gebhard Leopoldo G, Incicco J Jeremías, Smal Clara, Gallo Mariana, Gamarnik Andrea V, Kaufman Sergio B
Fundación Instituto Leloir-Consejo Nacional de Investigaciones Científicas y Técnicas, Ciudad Autónoma de Buenos Aires, C1405BWE, Argentina.
Instituto de Química y Fisicoquímica Biológicas and Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, C1113AAD, Argentina.
Nucleic Acids Res. 2014 Oct;42(18):11668-86. doi: 10.1093/nar/gku812. Epub 2014 Sep 15.
Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.
登革病毒非结构蛋白3（NS3）是一种多功能蛋白，由与蛋白酶结构域相连的超家族2 RNA解旋酶形成。在这项工作中，我们报告了体外实验的结果，这些实验旨在确定登革病毒NS3解旋酶（NS3h）的寡聚状态，并表征其与单链（ss）RNA相互作用的基本特性。脉冲场梯度核磁共振光谱用于确定NS3h的有效流体动力学半径，在不存在和存在ssRNA的情况下，该半径在很宽的蛋白质浓度范围内保持恒定。尺寸排阻色谱 - 静态光散射实验表明，即使在存在ssRNA的情况下，NS3h也以单体分子形式洗脱。使用不同长度的荧光素标记和未标记的ssRNA寡核苷酸通过定量荧光滴定研究了NS3h与ssRNA的结合，并分析了荧光素标记对相互作用参数的影响。基于大配体与一维晶格非特异性相互作用理论的统计热力学模型很好地描述了实验结果。我们发现，NS3h与ssRNA寡核苷酸和聚（A）的结合的特征在于最小和封闭结合位点大小均为10个核苷酸，以及相邻蛋白之间的弱正协同性。
