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-Mn2+, 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 Cu2+-binding paramagnetic probe that yields a much weaker PRE effect than EDTA-Mn2+ 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 phopshotransferase system, we have identified a minor species of excited-state complex with a similar to 4% population and exchanging with the stereospecific complex at similar to 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.