title: Practical considerations for investigation of protein conformational dynamics by15N R1ρrelaxation dispersion.
abstract: It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure ("excited states") has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by15N R1ρrelaxation dispersion. The schemes use selective Hartmann-Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713-721, 2005; Hansen et al. in J Am Chem Soc 131:3818-3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R1ρrelaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.
authors.affiliation: Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Erik
authors.initials: E
authors.lastname: Walinda
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Nishikyo-Ku, 615-8510, Kyoto, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Morimoto
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Nishikyo-Ku, 615-8510, Kyoto, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku Katsura, Nishikyo-Ku, 615-8510, Kyoto, Japan. sugase@moleng.kyoto-u.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Sugase
mesh_terms: D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D009587:Nitrogen Isotopes; D011506:Proteins
keywords: Cross-polarization; Heating compensation; Interleaved acquisition; Protein dynamics; Relaxation dispersion