Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes

[nmrlearner]

Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes

Abstract

Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient proteinâ??protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (15N or 13C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model proteinâ??protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence (1HN-Î?2-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time, unveiled the internal structural dynamics rearrangements of ubiquitin upon interaction. In addition to reducing the cost of sample preparation and speed up PRE measurements, by detecting the intra-molecular PRE this new strategy will make it possible to measure and calibrate inter-molecular distances more accurately for both symmetric and asymmetric proteinâ??protein complexes.



Source: Journal of Biomolecular NMR