Tuning the affinity for lanthanides of calcium binding proteins.
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.
Bertini
Ivano
I
Magnetic Resonance Center and Department of Chemistry, University of Florence, Sesto Fiorentino, Italy. bertini@cerm.unifi.it
Gelis
Ioannis
I
Katsaros
Nikolaos
N
Luchinat
Claudio
C
Provenzani
Alessandro
A
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D020832:EF Hand Motifs / Q000235:genetics; D028581:Lanthanoid Series Elements / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D002147:Calmodulin; D028581:Lanthanoid Series Elements; D011994:Recombinant Proteins; D002118:Calcium