Mekkattu Tharayil Sreelakshmi, Mahawaththa Mithun Chamikara, Loh Choy-Theng, Adekoya Ibidolapo, Otting Gottfried
ARC Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Canberra ACT 2601, Australia.
present address: Hangzhou Wayland Bioscience Co. Ltd, Hangzhou 310030, PR China.
Magn Reson (Gott). 2021 Jan 6;2(1):1-13. doi: 10.5194/mr-2-1-2021. eCollection 2021.
Pseudocontact shifts (PCSs) generated by paramagnetic lanthanide ions provide valuable long-range structural information in nuclear magnetic resonance (NMR) spectroscopic analyses of biological macromolecules such as proteins, but labelling proteins site-specifically with a single lanthanide ion remains an ongoing challenge, especially for proteins that are not suitable for ligation with cysteine-reactive lanthanide complexes. We show that a specific lanthanide-binding site can be installed on proteins by incorporation of phosphoserine in conjunction with other negatively charged residues, such as aspartate, glutamate or a second phosphoserine residue. The close proximity of the binding sites to the protein backbone leads to good immobilization of the lanthanide ion, as evidenced by the excellent quality of fits between experimental PCSs and PCSs calculated with a single magnetic susceptibility anisotropy ( tensor. An improved two-plasmid system was designed to enhance the yields of proteins with genetically encoded phosphoserine, and good lanthanide ion affinities were obtained when the side chains of the phosphoserine and aspartate residues are not engaged in salt bridges, although the presence of too many negatively charged residues in close proximity can also lead to unfolding of the protein. In view of the quality of the tensors that can be obtained from lanthanide-binding sites generated by site-specific incorporation of phosphoserine, this method presents an attractive tool for generating PCSs in stable proteins, particularly as it is independent of cysteine residues.
顺磁性镧系离子产生的伪接触位移（PCSs）在蛋白质等生物大分子的核磁共振（NMR）光谱分析中提供了有价值的远程结构信息，但用单个镧系离子对蛋白质进行位点特异性标记仍然是一个持续存在的挑战，特别是对于不适合与半胱氨酸反应性镧系配合物连接的蛋白质。我们表明，通过结合磷酸丝氨酸以及其他带负电荷的残基（如天冬氨酸、谷氨酸或第二个磷酸丝氨酸残基），可以在蛋白质上安装特定的镧系结合位点。结合位点与蛋白质主链的紧密接近导致镧系离子的良好固定，实验PCSs与用单个磁化率各向异性（张量）计算的PCSs之间的拟合质量极佳证明了这一点。设计了一种改进的双质粒系统以提高具有基因编码磷酸丝氨酸的蛋白质的产量，当磷酸丝氨酸和天冬氨酸残基的侧链不参与盐桥时，获得了良好的镧系离子亲和力，尽管在附近存在太多带负电荷的残基也会导致蛋白质解折叠。鉴于通过磷酸丝氨酸的位点特异性掺入产生的镧系结合位点可获得的张量质量，该方法为在稳定蛋白质中产生PCSs提供了一种有吸引力的工具,特别是因为它不依赖于半胱氨酸残基。
