An NMR Method for the Determination of Protein-Binding Interfaces Using Dioxygen-Induced Spin−Lattice Relaxation Enhancement
- 1 April 2005
- journal article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 127 (16) , 5826-5832
- https://doi.org/10.1021/ja047825j
Abstract
Using oxygen as a paramagnetic probe, researchers can routinely study topologies and protein-binding interfaces by NMR. The paramagnetic contribution to the amide 1H spin−lattice relaxation rates (R1P) have been studied for uniformly 2H,15N-labeled FB protein, a 60-residue three-helix bundle, constituting the B domain of protein A. Through TROSY versions of inversion−recovery experiments, R1P could be determined. R1P was then measured in the presence of a stoichiometric equivalent of an unlabeled Fc fragment of immunoglobulin (Ig) G, and the ratio of R1P of the FB−Fc complex to that of free FB [i.e., R1P(complex)/R1P(free)] was determined for each observable residue. Regions of helix I and helix II, which were previously known to interact with Fc, were readily identified as belonging to the binding interface by their characteristically reduced values of R1P(complex)/R1P(free). The method of comparing oxygen-induced spin−lattice relaxation rates of free protein and protein−protein complexes, to detect binding interfaces, offers greater sensitivity than chemical shift perturbation, while it is not necessary to heavily deuterate the labeled protein, as is the case in cross saturation experiments.Keywords
This publication has 24 references indexed in Scilit:
- Determination of the Interface of a Large Protein Complex by Transferred Cross-saturation MeasurementsJournal of Molecular Biology, 2002
- Discovering High-Affinity Ligands for Proteins: SAR by NMRScience, 1996
- Permeation Process of Small Molecules across Lipid Membranes Studied by Molecular Dynamics SimulationsThe Journal of Physical Chemistry, 1996
- Identification of the Contact Surface of a Streptococcal Protein G Domain Complexed with a Human Fc FragmentJournal of Molecular Biology, 1993
- Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra: Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filteringJournal of Molecular Biology, 1992
- NMR identification of protein surfaces using paramagnetic probesBiochemistry, 1990
- Sequential proton NMR assignments and secondary structure of the B domain of staphylococcal protein A: structural changes between the free B domain in solution and the Fc-bound B domain in crystalBiochemistry, 1990
- Studies of protein hydration in aqueous solution by direct NMR observation of individual protein-bound water moleculesJournal of the American Chemical Society, 1989
- The stereochemistry and dynamics of natural products and biopolymers from proton relaxation spectroscopy: spin-label delineation of inner and outer protons of gramicidin S including hydrogen bondsJournal of the American Chemical Society, 1982
- Detection of the helix-coil transition of polypeptides by ultrasonic absorption measurements in the megahertz range. Case of poly-L-glutamic acidJournal of the American Chemical Society, 1972