Gutenthaler Sophie M, Tsushima Satoru, Steudtner Robin, Gailer Manuel, Hoffmann-Röder Anja, Drobot Björn, Daumann Lena J
Department of Chemistry Ludwig-Maximilians-University Munich Butenandtstraße 5-13 81377 München Germany
Institute of Resource Ecology Helmholtz-Zentrum Dresden-Rossendorf e.V. Bautzner Landstraße 400 01328 Dresden Germany
Inorg Chem Front. 2022 Jun 30;9(16):4009-4021. doi: 10.1039/d2qi00933a. eCollection 2022 Aug 9.
Lanmodulin (LanM), a naturally lanthanide (Ln)-binding protein with a remarkable selectivity for Lns over Ca(ii) and affinities in the picomolar range, is an attractive target to address challenges in Ln separation. Why LanM has such a high selectivity is currently not entirely understood; both specific amino acid sequences of the EF-Hand loops and cooperativity effects have been suggested. Here, we removed the effect of cooperativity and synthesised all four 12-amino acid EF-Hand loop peptides, and investigated their affinity for two Lns (Eu(iii) and Tb(iii)), the actinide Cm(iii) and Ca(ii). Using isothermal titration calorimetry and time-resolved laser fluorescence spectroscopy (TRLFS) combined with parallel factor analysis, we show that the four short peptides behave very similarly, having affinities in the micromolar range for Eu(iii) and Tb(iii). Ca(ii) was shown not to bind to the peptides, which was verified with circular dichroism spectroscopy. This technique also revealed an increase in structural organisation upon Eu(iii) addition, which was supported by molecular dynamics simulations. Lastly, we put Eu(iii) and Cm(iii) in direct competition using TRLFS. Remarkably, a slightly higher affinity for Cm(iii) was found. Our results demonstrate that the picomolar affinities in LanM are largely an effect of pre-structuring and therefore a reduction of flexibility in combination with cooperative effects, and that all EF-Hand loops possess similar affinities when detached from the protein backbone, albeit still retaining the high selectivity for lanthanides and actinides over calcium.
镧调蛋白（LanM）是一种天然的镧系元素（Ln）结合蛋白，对Ln的选择性远高于Ca（ii），亲和力在皮摩尔范围内，是解决Ln分离挑战的一个有吸引力的目标。目前尚不完全清楚LanM为何具有如此高的选择性；有人提出这与EF-手型环的特定氨基酸序列和协同效应有关。在这里，我们消除了协同效应的影响，合成了所有四种12个氨基酸的EF-手型环肽，并研究了它们对两种Ln（Eu（iii）和Tb（iii））、锕系元素Cm（iii）和Ca（ii）的亲和力。使用等温滴定量热法和时间分辨激光荧光光谱法（TRLFS）结合平行因子分析，我们发现这四种短肽的行为非常相似，对Eu（iii）和Tb（iii）的亲和力在微摩尔范围内。结果表明Ca（ii）不与这些肽结合，这通过圆二色光谱法得到了验证。该技术还揭示了添加Eu（iii）后结构组织的增加，分子动力学模拟也证实了这一点。最后，我们使用TRLFS让Eu（iii）和Cm（iii）直接竞争。值得注意的是，发现对Cm（iii）的亲和力略高。我们的结果表明，LanM中的皮摩尔亲和力在很大程度上是预结构的作用，因此是结合协同效应导致的灵活性降低的结果，并且所有EF-手型环从蛋白质主链上分离后具有相似的亲和力，尽管对镧系元素和锕系元素的选择性仍然高于钙。
