NMR Spectroscopy for Monitoring Functional Dynamics in Solution

Abstract

Protein functional dynamics are defined as the atomic thermal fluctuations or the segmental motions that are essential for the function of a biomolecule. NMR is a very versatile technique that is well suited for obtaining quantitative information from these processes at atomic resolution and in multiple timescales. This article focuses on recent NMR developments to study functional dynamics, making special incidence in the experiments aimed for the characterisation of chemical–conformational exchange in the microsecond to millisecond timescale. In a second section, the novel‐solution NMR techniques that combine chemical exchange with saturation transfer experiments are addressed to finish with an overview on the dynamic information that can be extracted from residual dipolar couplings.

Key Concepts

  • NMR is well suited to study biomolecular dynamics at multiple timescales.
  • Many enzymatic reactions occur in the microsecond to millisecond timescale.
  • Relaxation dispersion experiments can extract quantitative information about the exchange process.
  • CEST and DEST experiments provide useful information on conformations that are invisible in the NMR spectrum.
  • Residual dipolar couplings contain information about spin dynamics that can be extracted and quantified.

Keywords: protein functional dynamics; NMR spectroscopy; residual dipolar couplings; CEST; DEST; conformational exchange; relaxation dispersion experiments; spin‐lock field

Figure 1. (a) Spin relaxation experiment cover most of the timescales where the effective protein internal (and overall) motions take place. Faster librations can be detected by nuclear spin relaxation measurements (R1, R2 and NOE) or by analysing the dynamic contribution of the residual dipolar couplings (RDCs). The line‐shape analysis and the relaxation dispersion experiments account for motions involving a larger number of atoms and are most relevant in biology. The slower motions can be characterised in NMR using EXSY and ZZ exchange experiments. (b) Owing to their intrinsic flexibility, proteins sample a range of thermodynamically accessible conformations within a hierarchy of timescales. The population of each state is determined by its Gibbs free energy, while the inter‐conversion between states is owed to the energy barriers that separate them. Three examples of the continuum of timescales are depicted in the figure for clarity: fast bond librations occurring in the picosecond (ps) to nanosecond (ns) timescale, segmental motions involving a larger number of atoms occurring in the microsecond (µs) to millisecond (ms) timescale and large domain rearrangements, occurring in slow timescales (s).
Figure 2. CPMG relaxation dispersion experiments for three different residues of the bacterial gene repressor YmoA. The transversal relaxation rate is measured as a function of the pulse train frequency. The solid lines correspond to the best fitting to the relaxation dispersion profile. Different residues show different sensitivities to the experiment and show no dispersion (red), moderate exchange contribution (blue) and a large contribution (black) of chemical exchange to line broadening.
Figure 3. Residual dipolar couplings. (a) Schematic representation of protein (red blob) in anisotropic alignment medium (vertical cylinders) with respect to the magnetic field 0. (b) Dynamics probed by RDC, the Azz, Ayy and Axx axes represents the alignment tensor for nuclear dipole pairs (bond vector with white and black balls) in a protein depicted in blue and the relative orientation of same dipole pairs in different alignment condition, where the protein is shown as red and golden yellow. The different accessible space available for the dipole pairs makes it feasible to obtain dynamics by RDC.
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Sivanandam, VN, and Millet, Oscar(Apr 2015) NMR Spectroscopy for Monitoring Functional Dynamics in Solution. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003104.pub2]