A paramagnetic probe to localize residues next to carboxylates on protein surfaces
A paramagnetic probe to localize residues next to carboxylates on protein surfaces
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DOI：
10.1007/s00775-002-0340-8
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发表时间：
2002-06-01
影响因子：
3
通讯作者：
Valensin, G
中科院分区：
文献类型：
--
作者：
Aime, S;D'Amelio, N;Valensin, G
研究表明，镧系元素的顺磁性可以用来获取蛋白质表面特定部分的信息。由于配位不饱和镧系配合物对供氧体具有很高的亲和力，羧酸基可以作为相互作用的优先目标。DO3A配体在这些研究中特别有用，因为它以七齿体的方式协调镧系元素，为外源供体留下两个位点。用高达200%的Gd(III)-DO3A络合物滴定n -15标记的样品蛋白calbindin D-9k（75个残基）的溶液，并使用反转回收N-15-H-1 HSQC实验来测量顺磁对酰胺质子纵向弛豫速率的贡献。利用弛豫数据作为距离约束来估计相互作用配合物的数量及其结合位点的占有率。在蛋白质表面发现了四个优先相互作用位点。对蛋白质表面各种羧酸侧链的检查表明，Gd(III)-DO3A优先与富含羧酸的区域相互作用，而不是与分离的羧酸相互作用，这表明一个Gd(III)-DO3A分子可能被两个羧酸基团螯合。因此，Gd(III)-DO3A是一种有价值的半选择性探针，用于探测蛋白质表面的负电荷簇。
It is shown that the paramagnetic properties of lanthanides can be exploited to obtain information on specific parts of a protein surface. Owing to the high affinity of coordinatively unsaturated lanthanide complexes for oxygen donors, carboxylate groups can be used as preferential targets for the interaction. The DO3A ligand is particularly useful in these studies, as it coordinates lanthanides in a heptadentate fashion, leaving two sites available for exogenous donors. A solution of a N-15-labeled sample protein, calbindin D-9k (75 residues), was titrated with up to 200% of Gd(III)-DO3A complex, and an inversion recovery N-15-H-1 HSQC experiment was used to measure the paramagnetic contributions to the longitudinal relaxation rates of the amide protons. Relaxation data were used as distance constraints to estimate the number of interacting complexes and the occupancies of their binding sites. Four preferential interaction sites on the protein surface are found. Inspection of the various carboxylate side chains on the surface of the protein indicates that Gd(III)-DO3A interacts preferentially with carboxylate-rich regions, rather than with isolated carboxylates, suggesting the possibility of chelation of one Gd(III)-DO3A molecule by two carboxylate groups. Gd(III)-DO3A is thus a valuable semi-selective probe for clusters of negative charges on the protein surface.