高分辨率质谱用于无标记,在不存在和存在氨基糖苷配体新霉素B的情况下,新霉素感应核糖开关适体结构域的CMC修饰的直接定位和相对定量,核糖霉素,和巴龙霉素.游离核糖开关的化学探测和MS数据显示,尿苷核碱基U7,U8,U13,U14,U18的溶剂高度暴露于拟议的内部和顶端环的一部分,但那些U10和U21作为建议的内部循环的一部分被发现是远低于预期的暴露。因此,与游离RNA相比,我们的数据与氨基糖苷复合物中核糖开关的拟议二级结构更吻合。对于新霉素B复合物中的核开关,核糖霉素,和巴龙霉素,我们发现了高度相似的CMC+修饰模式,并且与以前的NMR研究非常吻合。在不存在和存在氨基糖苷配体的情况下,化学探测和MS数据之间的差异是定量的,而不是定性的(i。Procedures.,相同的核碱基被标记,但在不同程度上),并且可以通过氨基糖苷结合稳定拟议的凸起和顶端环来合理化。我们的研究表明,化学探测和质谱可以提供重要的结构信息,并补充其他技术,如NMR光谱。
High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing
riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free
riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the
riboswitch in complexes with aminoglycosides than with that of free RNA. For the
riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our
study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.