Tuning the Affinity for Lanthanides of Calcium Binding Proteins

Abstract

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 (1)H-(15)N HSQC NMR spectra of the (15)N-enriched protein upon titration with Yb(3+). 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.

Experimentally exploring the conformational space sampled by domain reorientation in calmodulin

“…The plasmid pET16b-CaM was used to transform the BL21(DE3)gold Escherichia coli strain (Novagen) and transformants were used for the expression of unlabeled samples as previously described (29). 15 N-labeled N60D CaM was prepared as previously reported for unlabeled samples (29), except that immediately before induction the cells were gently pelleted and resuspended in M9 minimal medium supplemented with the appropriate isotopically labeled nitrogen (1. Bacterial cultures were induced with 0.5 mM isopropyl ␤-Dthiogalactoside, and the cells were further incubated at 37°C overnight.…”

“…The N60D mutant was found to increase the relative affinity of calcium for the second site of the N-terminal domain to an extent that permitted the preparation of well defined (CaLn) N (Ca 2 ) C CaM derivatives (29).…”

Examination of Matrix Metalloproteinase-1 in Solution

“…from the presence of paramagnetic metals, incorporated either in an existing metal binding site (36) or in a tag covalently bound to the protein (33). In the present case a lanthanide binding tag was used.…”

Loss of Protein Kinase Cδ/HuR Interaction Is Necessary to Doxorubicin Resistance in Breast Cancer Cell Lines

“…HuR mutants were generated by polymerase chain reaction (PCR) site-directed mutagenesis (QuikChange multisitedirected mutagenesis kit; Stratagene, La Jolla, CA) as previously described (Bertini et al, 2003) using as template pTrueOrf-HuR-MycHis (D' Agostino et al, 2013) and with the following primers: S221A (forward: 59-gcagagattcaggttcGCccccatgggcgtcg-39; reverse: 59-GACGC-CCATGGGGGCGAACCTGAATCTCTGC-39); and S318A (forward:: 59-ggacaaaatcttacaggttGccttcaaaaccaacaagtcc-39; reverse: 59-GGACT-TGTTGGTTTTGAAGGCAACCTGTAAGATTTTGTCC-39).…”