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A General Small-Angle X-ray Scattering-Based Screening Protocol Validated for Protein–RNA Interactions
ACS Combinatorial Science ( IF 3.903 ) Pub Date : 2018-03-19 00:00:00 , DOI: 10.1021/acscombsci.8b00007
Po-chia Chen 1 , Pawel Masiewicz 1 , Vladimir Rybin 1 , Dmitri Svergun 2 , Janosch Hennig 1
Affiliation  

We present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural information on biomolecular interactions independent of prior knowledge, so as to complement affinity-based screening and provide leads for further exploration. This protocol categorizes ligand titrations by computing pairwise agreement between curves, and separately estimates affinities by quantifying complex formation as a departure from the linear sum properties of solution SAXS. The protocol is validated by sparse sequence search around the native poly uridine RNA motifs of the two-RRM domain Sex-lethal protein (Sxl). The screening of 35 RNA motifs between 4 to 10 nucleotides reveals a strong variation of resulting complexes, revealed to be preference-switching between 1:1 and 2:2 binding stoichiometries upon addition of structural modeling. Validation of select sequences in isothermal calorimetry and NMR titration retrieves domain-specific roles and function of a guanine anchor. These findings reinforce the suitability of SAXS as a complement in lead identification.

中文翻译:

常规的小角度X射线散射为基础的筛选协议进行蛋白质-RNA相互作用的验证。

我们提出了一种利用小角度X射线散射(SAXS)来获得与生物分子相互作用有关的结构信息的筛查方案,该技术信息独立于先验知识,从而补充了基于亲和力的筛查并为进一步探索提供了线索。该协议通过计算曲线之间的成对一致性来对配体滴定进行分类,并通过量化复杂的形成量(与溶液SAXS的线性总和性质背离)来分别估计亲和力。通过稀疏序列搜索来验证该协议,该稀疏序列搜索是围绕两个RRM域性致死蛋白(Sx1)的天然聚尿苷RNA基序进行的。筛选4至10个核苷酸之间的35个RNA基序,揭示了所得复合物的强烈变化,显示出在添加结构模型后在1:1和2:2的结合化学计量之间进行了偏好转换。在等温量热法和NMR滴定法中验证选定序列的有效性,可检索鸟嘌呤锚蛋白的域特异性作用和功能。这些发现增强了SAXS作为铅鉴定中的补充的适用性。
更新日期:2018-03-19
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