PMID:16101285
| Citation |
Jensen, MR, Petersen, G, Lauritzen, C, Pedersen, J and Led, JJ (2005) Metal binding sites in proteins: identification and characterization by paramagnetic NMR relaxation. Biochemistry 44:11014-23 |
|---|---|
| Abstract |
A method is presented that allows the identification and quantitative characterization of metal binding sites in proteins using paramagnetic nuclear magnetic resonance spectroscopy. The method relies on the nonselective longitudinal relaxation rates of the amide protons and their dependence on the paramagnetic metal ion concentration and the pH, and on the three-dimensional structure of the protein. The method is demonstrated using Escherichia coli thioredoxin as a model protein and Ni(2+) as the paramagnetic metal ion. Through a least-squares analysis of the relaxation rates, it is found that Ni(2+) binds to a series of specific sites on the surface of thioredoxin. The strongest binding site is found near the N-terminus of the protein, where the metal ion is coordinated to the free NH(2) group of the N-terminal serine residue and the side chain carboxylate group of the aspartic acid residue in position 2. In addition, Ni(2+) binds specifically but more weakly to the surface-exposed side chain carboxylate groups of residues D10, D20, D47, and E85. |
| Links |
PubMed Online version:10.1021/bi0508136 |
| Keywords |
Amino Acids/chemistry; Amino Acids/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Models, Chemical; Models, Molecular; Nickel/chemistry; Nickel/metabolism; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Tertiary; Thioredoxins/chemistry; Thioredoxins/metabolism |
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