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Tuning the affinity for lanthanides of calcium binding proteins

机译：调整钙结合蛋白的镧系元素的亲和力

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The possibility of selectively substituting one or more lanthanides into the four canonical calcium binding sites of calcium-loaded vertebrate calmodulin (CaM) was investigated by monitoring changes in the H-1-N-15 HSQC NMR spectra of the N-15-enriched protein upon titration with Yb3+. The affinity of lanthanides for both N-terminal sites I and II is only moderately higher than that of calcium, and comparable with that of calcium for the two C-terminal sites. This situation induces binding of lanthanides to other noncanonical sites located at the interdomain linker, the N- and C-terminal ends, and at the inter-EF-hand linkers. Therefore, mutants were designed to alter the metal binding properties of ;calcium sites I (D22N, D24E), II (D58N, N60D, D58N-N60D), III (N97D), II-III (N60D-N97D), and IV (D129N), as well as of the inter-EF-hand linker of the N-terminal domain (N42K, T44K). The most striking effects were obtained for the N60D mutant at site II, as selective lanthanide binding is achieved even in the presence of excess calcium, and little or no population of the noncanonical sites occurs. Similar although less pronounced effects were observed for the N97D mutant. These findings allow us to better define some of the determinants of the relative affinities of calcium and lanthanides in CaM and, by extension, in other calcium binding proteins. Finally, by using CaM samples containing only three of the four calcium ions, it was possible to prepare well-defined Ca(3)Ln-CaM derivatives (Ln = Tb, Dy, Tm, and Yb), with interesting properties as NMR probes. [References: 55]
机译：通过监测N-15富集蛋白的H-1-N-15 HSQC NMR光谱的变化，研究选择性地将一种或多种镧系元素钙化成钙脊椎动物钙调钙调蛋白（CAM）的四个规范钙结合位点进行研究用YB3 +滴定。对于N-末端位点I和II的镧系元素的亲和力仅比钙高于钙，并且与两个C末端位点的钙相当。这种情况会使镧系元素与位于跨互联接头，N-和C末端的其他非甘露糖位点的结合，并且在EF手中的末端。因此，设计突变体以改变金属结合特性;钙位点I（D22N，D24E），II（D58N，N60D，D58N-N60D），III（N97D），III（N60D-N97D）和IV（ D129N），以及N末端域的EF手接头（N42K，T44K）。对于位点II的N60D突变体获得最引人注目的效果，因为即使在过量钙的存在下，也可以实现选择性镧系元素，并且少于或未发生非碳位点的群体。相似虽然对于N97D突变体观察到不太明显的效果。这些发现允许我们更好地定义凸轮中钙和镧系元素的相对亲和力的一些决定因素，并且通过延伸，在其他钙结合蛋白中。最后，通过使用仅含有四个钙离子中的三个的凸轮样品，可以制备明确定义的Ca（3）型Ln-Cam衍生物（Ln = Tb，Dy，Tm和Yb），其具有有趣的性质作为NMR探针。 [参考：55]

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