Investigation of slow molecular dynamics using R-CODEX

Abstract

A solid state NMR experiment is introduced for probing motions on the millisecond time scale, based on dephasing and refocusing ¹H–¹³C or ¹H–¹⁵N dipolar couplings. The method is related to the previously described Centerband-Only Detection of Exchange or CODEX experiment. The use of an R-type dipolar recoupling sequence takes advantage of the strong ¹H–¹³C or ¹H–¹⁵N dipolar coupling, while suppressing the effect of ¹H–¹H homonuclear coupling. This approach paves the way to detect both the correlation time and reorientational angle of the dynamics in fully protonated samples. The performance of this pulse sequence is demonstrated using imidazole methyl sulfonate.

Introduction

Molecular and conformational dynamics on the millisecond scale play important roles in protein function [1], [2], [3]. NMR methods provide the opportunity to probe the correlation time and the amplitude of a dynamical process in a site-specific fashion, which has the possibility to provide significant insights into protein function. For example, solution NMR studies, such as CPMG and R₁ρ experiments, can provide site-specific correlation times and populations describing the dynamics of proteins in solution [4], [5]. In some cases, the amplitude of the motion can also be obtained quantitatively from for example residual dipolar coupling (RDC) experiments, and insights about the structures of the two sites can be obtained through interpreting chemical shifts [6].

Solid state NMR methods can significantly broaden the range of systems amenable to this kind of analysis, and can be sensitive on a range of timescales. On the scale of milliseconds, seconds for example, a family of experiments known as Centerband-Only Detection of Exchange Experiments (CODEX) [7], [8], [9], [10], [11] exploit anisotropic interactions such as the chemical shift anisotropy (CSA), to encode the orientation of a site and provide the means to detect changes in the orientation, and obtain both the correlation time and reorientational angle. To interpret the data it is necessary to know the principal values and orientations of the CSA, which can be complex for systems like proteins [12], [13]. In addition, most of the CSA tensors are non-uniaxial, which also complicates the analysis.

Therefore, experiments based on heteronuclear dipolar coupling are an attractive alternative. The heteronuclear dipolar coupling is strictly uniaxial, and has a relatively fixed coupling constant with the principal component D_zz along the chemical bond. Schaefer et al. used CODEX with and without ¹³C–¹⁹F dipolar cancelation and demonstrated the presence of a large amplitude motion of a fluorinated aromatic ring [14]. Recently, Dipolar CODEX experiments [15] based on recoupling, ¹³C–¹⁵N dipolar couplings, using a rotational echo double resonance (REDOR) [16] pulse sequence element, have been reported, and other experiments that utilize ¹H–¹⁵N dipolar couplings have been implemented with applications on a protein [17]. The ¹³C–¹⁵N Dipolar CODEX requires a long dephasing time due to the weak ¹³C–¹⁵N dipolar interaction, resulting in potential loss of signal. The ¹H–¹⁵N Dipolar CODEX experiments had potential artifacts from ¹H homonuclear coupling and therefore require perdeuteration of the protein.

In this paper, we report a R-CODEX experiment, which recouples the ¹H−¹³C or ¹H–¹⁵N dipolar couplings via the $R{18}_1^7$ [18] recoupling element, a R-type sequence based on symmetry principles [19]. The π flip motion of the imidazolium ring in imidazolium methyl sulfonate was studied with this method to demonstrate properties of the pulse sequence. The results are promising for the detection of protein dynamics in the future. We discuss the advantages of this scheme in terms of experimental results, numerical simulations and theoretical considerations.