Metal Binding Sites in Proteins: Identification and Characterization by Paramagnetic NMR Relaxation
- 28 July 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 44 (33), 11014-11023
- https://doi.org/10.1021/bi0508136
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 Ni2+ as the paramagnetic metal ion. Through a least-squares analysis of the relaxation rates, it is found that Ni2+ 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 NH2 group of the N-terminal serine residue and the side chain carboxylate group of the aspartic acid residue in position 2. In addition, Ni2+ binds specifically but more weakly to the surface-exposed side chain carboxylate groups of residues D10, D20, D47, and E85.Keywords
This publication has 19 references indexed in Scilit:
- Paramagnetic NMR study of Cu2+ -IDA complex localization on a protein surface and its application to elucidate long distance informationFEBS Letters, 2004
- Validation of Protein Structure from Anisotropic Carbonyl Chemical Shifts in a Dilute Liquid Crystalline PhaseJournal of the American Chemical Society, 1998
- Determination of the Magnetic Axes of Cobalt(II) and Nickel(II) Azurins from 1H NMR Data: Influence of the Metal and Axial Ligands on the Origin of Magnetic Anisotropy in Blue Copper ProteinsBiochemistry, 1998
- The Solution Structure Refinement of the Paramagnetic Reduced High‐Potential Iron‐Sulfur Protein I from Ectothiorhodospira Halophila by Using Stable Isotope Labeling and Nuclear RelaxationEuropean Journal of Biochemistry, 1996
- MOLMOL: A program for display and analysis of macromolecular structuresJournal of Molecular Graphics, 1996
- The Design of Metal-Binding Sites in ProteinsAnnual Review of Biophysics, 1993
- The prediction and characterization of metal binding sites in proteinsProtein Engineering, Design and Selection, 1993
- Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxinFEBS Letters, 1991
- Crystal structure of thioredoxin from Escherichia coli at 1.68 Å resolutionJournal of Molecular Biology, 1990
- Perturbation of the PMR spectrum of lysozyme by Co+2Biochemical and Biophysical Research Communications, 1969