title: Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin.
abstract: To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.
authors.affiliation: Department of Chemistry, Center of Drug Design, Georgia State University, University Plaza, Atlanta, GA 30303, USA.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Ye
authors.forename: Hsiau-Wei
authors.initials: HW
authors.lastname: Lee
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D066298:In Vitro Techniques; D028581:Lanthanoid Series Elements / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D028581:Lanthanoid Series Elements; D011994:Recombinant Proteins; D002118:Calcium