Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups

Abstract

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for (1)H spins in methyl (13)CHD2 groups, which improves the characterization of fast exchange processes. The influence of (1)H-(1)H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R 1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different (1)H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any (1)H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by (1)H and (13)C CPMG experiments. The present R 1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.

Conformational heterogeneity of the calmodulin binding interface

“…In the absence of Ca 2+ , exchange with the holo-like state occurs with a k ex of ∼26 μs, within the range of 18–150 μs that has been experimentally determined for the isolated C-terminal domain ( Fig. 2a ) 30 33 34 35 36 37 . Transitions to the holo-like state coincide with aromatic stacking and formation of the second Ca 2+ -binding site, as assessed by inter-residue distances between Phe92 and Phe141 and between the Ca 2+ ligands Asp129 and Asp133, respectively.…”

Visualizing transient dark states by NMR spectroscopy

“…2009; Lundstrom & Akke, 2005; Weininger et al . 2013). The remainder of this section, however, focuses on CPMG relaxation dispersion.…”

Capturing Excited States in the Fast‐Intermediate Exchange Limit in Biological Systems Using 1H NMR Spectroscopy

“…We present results obtained for different types of 1 H spin systems, specifically H5, H1′, and H8 in RNA, and Hα positions in proteins (Figure A,B), without the need for selective 13 C or 2 H labelling . The pulse sequence (Figure ) is built on ideas from Lundström, Weininger, Eichmüller, and Hansen …”