Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University, Columbus, OH 43210, USA.
J Mol Biol. 2022 Apr 15;434(7):167480. doi: 10.1016/j.jmb.2022.167480. Epub 2022 Feb 14.
FraR, a transcriptional repressor, was postulated to regulate the metabolism of the Amadori compound fructose-asparagine (F-Asn) in the foodborne pathogen Salmonella enterica. Here, the DNA- and inducer-binding affinities and stoichiometries of FraR were determined and cross-validated by electrophoretic mobility-shift assays (EMSAs) and online buffer exchange coupled to native mass spectrometry (OBE-nMS). We demonstrate the utility of OBE-nMS to characterize protein and protein-DNA complexes that are not amenable to offline exchange into volatile buffers. OBE-nMS complemented EMSAs by revealing that FraR binds to the operator DNA as a dimer and by establishing 6-phosphofructose-aspartate as the inducer that weakens DNA binding by FraR. These results provide insights into how FraR regulates the expression of F-Asn-catabolizing enzymes and add to our understanding of the intricate bacterial circuitry that dictates utilization of diverse nutrients.
FraR 是一种转录抑制剂，据推测它可以调节食源性病原体沙门氏菌中 Amadori 化合物果糖-天冬酰胺（F-Asn）的代谢。在这里，通过电泳迁移率变动分析（EMSA）和在线缓冲交换结合天然质谱（OBE-nMS）确定 FraR 的 DNA 和诱导剂结合亲和力和化学计量比，并进行交叉验证。我们展示了 OBE-nMS 在表征不适于离线交换到挥发性缓冲液的蛋白质和蛋白质-DNA 复合物方面的应用。OBE-nMS 通过揭示 FraR 以二聚体形式与操纵子 DNA 结合，并确定 6-磷酸果糖-天冬氨酸作为诱导剂，从而削弱 FraR 对 DNA 的结合，补充了 EMSA 的结果。这些结果深入了解 FraR 如何调节 F-Asn 分解代谢酶的表达，并增加了我们对决定利用各种营养物质的复杂细菌电路的理解。
