Keizers Peter H J, Saragliadis Athanasios, Hiruma Yoshitaka, Overhand Mark, Ubbink Marcellus
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Post Office Box 9502, 2300 RA Leiden, The Netherlands.
J Am Chem Soc. 2008 Nov 5;130(44):14802-12. doi: 10.1021/ja8054832. Epub 2008 Oct 1.
Immobilized lanthanide ions offer the opportunity to refine structures of proteins and the complexes they form by using restraints obtained from paramagnetic NMR experiments. We report the design, synthesis, and spectroscopic evaluation of the lanthanide chelator, Caged Lanthanide NMR Probe 5 (CLaNP-5) readily attachable to a protein surface via two cysteine residues. The probe causes tunable pseudocontact shifts, alignment, paramagnetic relaxation enhancement, and luminescence, by chelating it to the appropriate lanthanide ion. The observation of single shifts and the finding that the magnetic susceptibility tensors obtained from shifts and alignment analyses are highly similar strongly indicate that the probe is rigid with respect to the protein backbone. By placing the probe at various positions on a model protein it is demonstrated that the size and orientation of the magnetic susceptibility tensor of the probe are independent of the local protein environment. Consequently, the effects of the probe are readily predictable using a protein structure only. These findings designate CLaNP-5 as a protein probe to deliver unambiguous high quality structural restraints in studies on protein-protein and protein-ligand interactions.
固定化镧系离子为通过顺磁核磁共振实验获得的约束条件来优化蛋白质及其形成的复合物的结构提供了机会。我们报告了镧系螯合剂“笼状镧系核磁共振探针5（CLaNP - 5）”的设计、合成及光谱评估，该探针可通过两个半胱氨酸残基轻松连接到蛋白质表面。通过将该探针螯合到合适的镧系离子上，它会引起可调谐的赝接触位移、取向、顺磁弛豫增强和发光现象。单位移的观察结果以及从位移和取向分析中获得的磁化率张量高度相似这一发现，有力地表明该探针相对于蛋白质主链是刚性的。通过将探针放置在模型蛋白质的不同位置，证明了探针的磁化率张量的大小和取向与局部蛋白质环境无关。因此，仅使用蛋白质结构就可以很容易地预测探针的作用效果。这些发现表明CLaNP - 5是一种蛋白质探针，可在蛋白质 - 蛋白质和蛋白质 - 配体相互作用的研究中提供明确的高质量结构约束条件。
