Modern Technologies of Solution Nuclear Magnetic Resonance Spectroscopy for Three-dimensional Structure Determination of Proteins Open Avenues for Life Scientists

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for structural studies of chemical compounds and biomolecules such as DNA and proteins. Since the NMR signal sensitively reflects the chemical environment and the dynamics of a nuclear spin, NMR experiments provide a wealth of structural and dynamic information about the molecule of interest at atomic resolution. In general, structural biology studies using NMR spectroscopy still require a reasonable understanding of the theory behind the technique and experience on how to recorded NMR data. Owing to the remarkable progress in the past decade, we can easily access suitable and popular analytical resources for NMR structure determination of proteins with high accuracy. Here, we describe the practical aspects, workflow and key points of modern NMR techniques used for solution structure determination of proteins. This review should aid NMR specialists aiming to develop new methods that accelerate the structure determination process, and open avenues for non-specialist and life scientists interested in using NMR spectroscopy to solve protein structures.

Keywords: Automation; Nuclear magnetic resonance (NMR) spectroscopy; Protein; Solution NMR; Structure determination; Validation.

Fig. 1

Workflow of protein structure determination by solution NMR spectroscopy. (A) First, the target protein is uniformly labeled with NMR active isotopes (¹³C and ¹⁵N) and purified by chromatography processes. In many cases, it is essential to optimize the solution composition of the NMR sample (e.g., buffer, pH, type and concentration of salt, and other additives to prevent aggregation and/or to improve thermal stability of the target protein) and NMR parameters (e.g., sample temperature during NMR data collection, strength of static magnetic field, pulse sequences, inversion recovery delay) prior to collecting all the multidimensional heteronuclear NMR experiments. (B) Generally, various sample conditions and NMR parameters are verified by measuring 2D ¹H–¹⁵N HSQC NMR spectra and/or 2D projection spectra of 3D HNCACB (e.g., 2D HN(CA)CB) until optimal conditions are found. If sample/NMR data collection optimization is performed by only ¹⁵N-based NMR experiments, only a ¹⁵N-labeled target protein (without ¹³C-labeling) is required. (C) The NMR signals of all ¹H, ¹³C and ¹⁵N nuclei are assigned by analyzing multidimensional heteronuclear NMR spectra using NMR software that facilitates assignment of the signals. The right lower panel of this figure is a screenshot of a NMR signal assignment using the Kujira and MagRO software, developed by Prof. Naohiro Kobayashi (Osaka University, Japan), which is available at PDBj-BMRB http://bmrbdep.pdbj.org/en/nmr_tool_box/magro_nmrview.html. (D) After completing the assignment process, the solution structures of the target protein are determined by performing simulated annealing using NMR-based restraints such as ¹H–¹H distances and dihedral angles derived from NOEs and chemical shifts, respectively. (E) Finally, the precision and the accuracy of the determined structure are verified statistically and experimentally (e.g., by analyzing the Ramachandran plot and measuring RDCs of the target protein, respectively).