Ching H Y Vincent, Mascali Florencia C, Bertrand Hélène C, Bruch Eduardo M, Demay-Drouhard Paul, Rasia Rodolfo M, Policar Clotilde, Tabares Leandro C, Un Sun
Institute for Integrative Biology of the Cell (I2BC), Department of Biochemistry, Biophysics and Structural Biology, Université Paris-Saclay, CEA, CNRS UMR 9198 , F-91191 Gif-sur-Yvette, France.
Instituto de Biología Molecular y Celular de Rosario; Área Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario , 2000 Rosario, Argentina.
J Phys Chem Lett. 2016 Mar 17;7(6):1072-6. doi: 10.1021/acs.jpclett.6b00362. Epub 2016 Mar 8.
A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 μM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements.
一种能够由天然可得成分自组装而成的基因编码顺磁自旋标记物，将为通过电子顺磁共振（EPR）研究蛋白质的细胞内结构和相互作用提供一种手段。在此，我们展示了对由与组氨酸序列（即所谓的His标签）配位的Mn(II)离子组成的自旋标记物进行脉冲电子-电子双共振（DEER）测量，His标签是通过基因工程普遍添加以促进蛋白质纯化的。尽管His标签对Mn(II)的亲和力较低（800 μM），但即使在细胞预期的浓度下，与Mn(II)结合的His标签也能产生易于检测的DEER时间轨迹。我们能够准确确定已知结构的刚性螺旋聚脯氨酸肽末端以及完全在细胞内合成的三螺旋束末端的两个His标签Mn(II)自旋标记物之间的距离。这种方法不仅大大简化了标记过程，而且代表了朝着使用自组装金属自旋标记物进行细胞内距离测量迈出的第一步。
