ih-RIDME: a pulse EPR experiment to probe the heterogeneous nuclear environment

Abstract. Intermolecular hyperfine relaxation-induced dipolar modulation enhancement experiment (ih-RIDME) is a pulse EPR experiment that can be used to probe the properties of a nuclear spin bath in the vicinity of an unpaired electron. The underlying mechanism is the hyperfine spectral diffusion of the electron spin during the mixing block. A quantitative description of the diffusion kinetics being applied to establish the ih-RIDME data model allows to extend this method for systems with heterogeneous nuclear arrangements assuming a distribution of the local nuclear densities. The heterogeneity can stem from the solvent or the intrinsic nuclei of structurally flexible (macro)molecule. Therefore, the fitted distribution function can further serve for heterogeneity characterization, quantification and structure-based analysis. Here, we present a detailed introduction to the principles of the ih-RIDME application to heterogeneous systems. We discuss the spectral resolution, determination of the spectral diffusion parameters and influence of noise in the experimental data. We further demonstrate the application of the ih-RIDME method to a model spin-labeled macromolecule with unstructured domains. The fitted distribution of local proton densities was reproduced with the help of the Monte-Carlo-generated conformational ensemble. Finally, we discuss several pulse sequences exploiting the HYperfine Spectral Diffusion Echo MOdulatioN (HYSDEMON) effect with an improved signal-to-noise ratio.