Lanmodulin peptides – unravelling the binding of the EF-Hand loop sequences stripped from the structural corset. Issue 16 (30th June 2022)

• Record Type: Journal Article
• Authors: Gutenthaler, Sophie M.; Tsushima, Satoru; Steudtner, Robin; Gailer, Manuel; Hoffmann-Röder, Anja; Drobot, Björn; Daumann, Lena J.
• Is Part Of: Inorganic chemistry frontiers. Volume 9:Issue 16(2022)
• Page Start: 4009
• Page End: 4021
• Publication Date: 2022-06-30
• Subjects: Chemistry, Inorganic -- Periodicals; 546.05
• DOI: 10.1039/d2qi00933a
• Languages: English
• ISSNs: 2052-1553

Abstract

Abstract : Taking a closer look at Lanmodulin's remarkable selectivity for lanthanides (Ln) over Ca(ii ) and high Ln/actinide affinities on the amino acid level by investigating the four binding-loops as peptides with Ca(ii ), Eu(iii ), Tb(iii ) and Cm(iii ). Abstract : 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.