Prudêncio Miguel, Rohovec Jan, Peters Joop A, Tocheva Elitza, Boulanger Martin J, Murphy Michael E P, Hupkes Hermen-Jan, Kosters Walter, Impagliazzo Antonietta, Ubbink Marcellus
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Chemistry. 2004 Jul 5;10(13):3252-60. doi: 10.1002/chem.200306019.
A lanthanide complex, named CLaNP (caged lanthanide NMR probe) has been developed for the characterisation of proteins by paramagnetic NMR spectroscopy. The probe consists of a lanthanide chelated by a derivative of DTPA (diethylenetriaminepentaacetic acid) with two thiol reactive functional groups. The CLaNP molecule is attached to a protein by two engineered, surface-exposed, Cys residues in a bidentate manner. This drastically limits the dynamics of the metal relative to the protein and enables measurements of pseudocontact shifts. NMR spectroscopy experiments on a diamagnetic control and the crystal structure of the probe-protein complex demonstrate that the protein structure is not affected by probe attachment. The probe is able to induce pseudocontact shifts to at least 40 A from the metal and causes residual dipolar couplings due to alignment at a high magnetic field. The molecule exists in several isomeric forms with different paramagnetic tensors; this provides a fast way to obtain long-range distance restraints.
一种名为CLaNP（笼形镧系元素核磁共振探针）的镧系元素配合物已被开发出来，用于通过顺磁核磁共振光谱法表征蛋白质。该探针由镧系元素与二乙三胺五乙酸（DTPA）的衍生物螯合而成，带有两个硫醇反应性功能基团。CLaNP分子通过两个经工程改造、暴露于表面的半胱氨酸残基以双齿方式连接到蛋白质上。这极大地限制了金属相对于蛋白质的动力学，并能够测量伪接触位移。对抗磁对照物进行的核磁共振光谱实验以及探针 - 蛋白质复合物的晶体结构表明，蛋白质结构不受探针连接的影响。该探针能够在距金属至少40埃处诱导伪接触位移，并由于在高磁场下的取向而产生残余偶极耦合。该分子以几种具有不同顺磁张量的异构体形式存在；这提供了一种获得长程距离限制的快速方法。
