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.
Highlights
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for structural studies of chemical compounds and biomolecules such as DNA and proteins
When only a minimal number of NOE signals are observed owing to the poor character of the target protein, e.g. aggregation or signal line broadening owing to averaging of multiple conformation caused by fluctuation in an intermediate-slow NMR time scale regime, long-range distance information can be collected, which may complement the shortage of NOEbased distance restraints, by protein labeling with paramagnetic metal ions or radicals to measure the paramagnetic relaxation enhancement (PRE) effect [105] or the pseudo-contact shift (PCS) [106,107]
Solution NMR spectroscopy is a popular method to determine the tertiary structure of proteins
Summary
The standard approach to protein structure determination by solution NMR comprises several steps: (i) preparation of a protein sample labeled with stable NMR spin-1/2 isotopes, 13C and 15N; (ii) acquisition of NMR spectra; (iii) data processing and assignments of signals; (iv) generation of a chemical shift table that gives the signal assignments derived from the analysis of NMR spectra such that the analyst or an automated program can subsequently assign NOE signals to generate. Distance or dihedral angle restraints; and (v) simulated annealing (SA) by simplified molecular dynamics calculations with the NMR-based restraints are used for initial structure modeling to obtain an ensemble of structures that satisfy the experimentally determined constraints. These SA calculations are followed by molecular dynamics (MD) simulation with explicit or implicit water using a more sophisticated force field. (vi) The final stage of the workflow is validation of the precision and accuracy of the determined structure (Fig. 1) Details of these steps are explained in the subsections below.
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