title: Characterizing dynamic protein-protein interactions using differentially scaled paramagnetic relaxation enhancement.
abstract: Paramagnetic relaxation enhancement (PRE) is a powerful NMR technique that allows direct visualization of minor species. The PRE is obtained by conjugating a paramagnetic probe, such as EDTA-Mn(2+), at a specific cysteine residue. For a fast exchange between major and minor species, the observed PRE rate approaches population-weighted average of PRE values for both states. We have employed a tripeptide of Cu(2+)-binding paramagnetic probe that yields a much weaker PRE effect than EDTA-Mn(2+) does. We show that by using two probes of different paramagnetic strengths attached at the same site, the relative population and exchange time scale can be extracted, providing that the dynamic event occurs in the second to millisecond regime. Hence, this improved PRE scheme, differentially scaled paramagnetic relaxation enhancement (DiSPRE), permits both temporal and spatial characterization of a dynamic system. When applying the DiSPRE scheme to reassess the weak interactions between the N-terminal domain of enzyme I and phosphocarrier protein (HPr) from the bacterial phosphotransferase system, we have identified a minor species of excited-state complex with a approximately 4% population and exchanging with the stereospecific complex at approximately 1100 s(-1). Such species is distinct from other encounter complexes previously characterized and is likely a result of promiscuity of the HPr binding interface.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Yu
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Volkov
authors.forename: Chun
authors.initials: C
authors.lastname: Tang
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins