Supplementary Information files for A chiral lanthanide tag for stable and rigid attachment to single cysteine residues in proteins for NMR, EPR and time-resolved luminescence studies

Supplementary Information files for A chiral lanthanide tag for stable and rigid attachment to single cysteine residues in proteins for NMR, EPR and time-resolved luminescence studies<br>A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron–electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

用于核磁共振（NMR）、电子顺磁共振（EPR）与时间分辨发光研究的、可稳定刚性结合至蛋白质单半胱氨酸残基的手性镧系元素标记物补充信息文件。可位点特异性结合至蛋白质的镧系元素结合标记物，为通过多种光谱技术（包括核磁共振、电子顺磁共振与时间分辨发光光谱）研究蛋白质提供了手段。本研究报道了一种新型稳定手性三价镧系（LnIII）标记物，命名为C12，它可与半胱氨酸残基发生自发且定量的反应，形成稳定的硫醚键。该标记物的合成方案相对简便，且便于储存与运输。其对硒代半胱氨酸展现出显著提升的反应活性，为在含半胱氨酸残基的蛋白质中选择性标记硒代半胱氨酸提供了可行途径。将三价铽（TbIII）或三价铥（TmIII）离子负载至C12标记物后，可在蛋白质核磁共振光谱中便捷地产生伪接触位移（PCS）。该标记物可在镧系元素与蛋白质之间形成相对刚性的连接臂，这有利于通过单一磁各向异性张量对伪接触位移进行解析，同时适用于双电子-电子共振实验中的距离分布测量。当与半胱氨酸或其他巯基化合物发生反应后，三价铽配合物的发光量子产率可提升100倍，可为时间分辨荧光共振能量转移（FRET）分析与酶反应监测提供高灵敏的开启型发光探针。