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.
Maximum occurrence analysis of protein conformations for different distributions of paramagnetic metal ions within flexible two-domain proteins
Maximum occurrence analysis of protein conformations for different distributions of paramagnetic metal ions within flexible two-domain proteins
Publication year: 2011
Source: Journal of Magnetic Resonance, Available online 30 December 2011</br>
Claudio*Luchinat, Malini*Nagulapalli, Giacomo*Parigi, Luca*Sgheri</br>
Multidomain proteins are composed of rigid domains connected by (flexible) linkers. Therefore, the domains may experience a large degree of reciprocal reorientation. Pseudocontact shifts and residual dipolar couplings arising from one or more paramagnetic metals successively...
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Authors: Jensen MR, Led JJ
Four different approaches for determining the electron relaxation rates in paramagnetic metallo-proteins are investigated, using a paramagnetic Ni2+ complex of a protein as an example. All four approaches rely on the...
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[NMR paper] Metal-ligand interactions in perturbed blue copper sites: a paramagnetic (1)H NMR stu
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Authors: Fernández CO, Niizeki T, Kohzuma T, Vila AJ
Pseudoazurin is an electron transfer copper protein, a member of the cupredoxin family. The protein is frequently found in denitrifying bacteria, where it is the electron donor of nitrite reductase. The copper at...
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[NMR paper] Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectr
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J Mol Biol. 2002 Sep 13;322(2):425-40
Authors: Rubin SM, Lee SY, Ruiz EJ, Pines A, Wemmer DE
Xenon-binding sites in proteins have led to a number of applications of xenon in biochemical and structural studies. Here we further develop the utility of 129Xe NMR in characterizing specific xenon-protein interactions. The sensitivity of the 129Xe...
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Lead (Pb), mercury (Hg), and cadmium (Cd) are toxic and interfere with...
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[NMR paper] Identification of the ribosome binding sites of translation initiation factor IF3 by
Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy.
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Titrations of Escherichia coli translation initiation...
Metal binding sites in proteins: identification and characterization by paramagnetic NMR relaxation.
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