An NMR method for the determination of protein-binding interfaces using dioxygen-induced spin-lattice relaxation enhancement.
Using oxygen as a paramagnetic probe, researchers can routinely study topologies and protein-binding interfaces by NMR. The paramagnetic contribution to the amide (1)H spin-lattice relaxation rates (R(1)(P)) have been studied for uniformly (2)H,(15)N-labeled FB protein, a 60-residue three-helix bundle, constituting the B domain of protein A. Through TROSY versions of inversion-recovery experiments, R(1)(P) could be determined. R(1)(P) was then measured in the presence of a stoichiometric equivalent of an unlabeled Fc fragment of immunoglobulin (Ig) G, and the ratio of R(1)(P) of the FB-Fc complex to that of free FB [i.e., R(1)(P)(complex)/R(1)(P)(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 R(1)(P)(complex)/R(1)(P)(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.
Sakakura
M
M
Contribution from the Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Noba
S
S
Luchette
P A
PA
Shimada
I
I
Prosser
R S
RS
D003903:Deuterium; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D009587:Nitrogen Isotopes; D011522:Protons; D013205:Staphylococcal Protein A; D003903:Deuterium; D010100:Oxygen