Elucidating transient macromolecular interactions using paramagnetic relaxation enhancement

Abstract

Recent advances in the use of paramagnetic relaxation enhancement (PRE) in structure refinement and in the analysis of transient dynamic processes involved in macromolecular complex formation are presented. In the slow exchange regime, we show, using the SRY/DNA complex as an example, that the PRE provides a powerful tool that can lead to significant increases in the reliability and accuracy of NMR structure determinations. Refinement necessitates the use of an ensemble representation of the paramagnetic center and a model free extension of the Solomon-Bloembergen equations. In the fast exchange regime, the PRE provides insight into dynamic processes and the existence of transient, low population intermediate species. The PRE allows one to characterize dynamic non-specific binding of a protein to DNA; to directly demonstrate that the search process whereby a transcription factor locates its specific DNA target site involves both intramolecular (sliding) and intermolecular (hopping and intersegment transfer) translocation; and to detect and visualize the distribution of an ensemble of transient encounter complexes in protein-protein association.

Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR

“…In the PRE experiments, each of the residues Glu19, Arg23, Ala29, Ala32, Pro38, and Pro44 in HMGN2 was mutated to Cys individually and linked by a disulfide bond to the paramagnetic compound S-Methanethiosulfonylcysteaminyl (MTS)-EDTAMn 2þ . Binding of paramagnetic spin-labeled HMGN2 reduces peak intensities of the methyl groups in the nucleosome in a manner depending on their distances to the paramagnetic center (21). The N-terminal spin labels (Glu19, Arg23, Ala29, and Ala32) all affect methyl groups near the acidic patch, confirming the chemical shift perturbation results.…”

From a structural average to the conformational ensemble of a DNA bulge

“…X-ray interferometry readily provides information about translational displacements, which are difficult to assess with RDC measurements (47), as well as angular movements, and it is more straightforward to extend to larger structures (22,24) and other classes of macromolecules. However, X-ray interferometry is limited in detecting rare conformers (17,18,23) so that techniques that can trap or assess rare excursions, such as NMR relaxation dispersion (48), paramagnetic relaxation enhancement (49), hydrogen/deuterium exchange (50), and cyclization (51), are powerful complements to X-ray interferometry. Full atomic-level resolution of conformational ensembles and free-energy landscapes of macromolecules will require continued synergy between the development of X-ray interferometry, RDC measurements, and other experimental techniques as well as MD and other computations approaches.…”

Bax activation by Bim?

“…This method has recently been shown to be useful for the characterization of low affinity interactions in a variety of systems. 28,29 Use of PRE allowed for residues on Bax in the vicinity of the paramagnetic label on the peptide to be identified. Placing the label at the N terminus of the stapled BimBH3 peptide identified residues within a6, a1, a1-a2 loop and a4 of Bax.…”