Table 1.

General outline of different NMR methodologies employed to quantify protein dynamics on a broad range of time frames. Partly adapted from ref. ¹⁰.

Method	Description	Information provided	Time Scale of Protein Motions Investigated	Exchange Ratea (kex) Characterized	Selected References
H/D Exchange	Detection of hydrogen exchange with a fully deuterated solvent: disappearance of amide proton signals over time	Solvent accessibility, hydrogen bond strength, slow conformational exchange	Seconds-minutes		91,92
Real-Time NMR (RT NMR)	Direct detection of dynamic processes by quantifying the time-dependence of NMR signal intensity after a perturbation of the system	Protein folding, solvent hydrogen-exchange, slow conformational exchange	Seconds	kex < 1 s−1 kex ≪ Δω	93,94
ZZ-exchange (Exchange Spectroscopy; EXSY)	Simultaneous determination of 15N longitudinal relaxation and chemical exchange during a mixing delay	Slow conformational exchange such as domain movement, ligand binding and release, topological interconversion of secondary structure, cis-trans isomerization	Milliseconds-seconds	kex ≈ 0.2–100 s−1 kex ≪ Δω	95,96
Chemical-and Dark-state Exchange Saturation Transfer (CEST/DEST)	Saturation transfer by chemical exchange between invisible (minor, excited) state and visible (major, ground) state	Information on invisible states, populations of the states, exchange rates and slow-intermediate conformational exchange such as small domain movements	Milliseconds	kex ≈ 10–100 s−1 kex ≤ Δω	6,28
Carr-Purcell Meiboom-Gill Relaxation Dispersion (CPMG RD)	Attenuation of chemical exchange broadening by applying refocusing pulses: reduce transverse relaxation R2 sensitive to motions	Kinetic, thermodynami c, and structural information for intermediate-fast exchange processes such as side chain reorientation, loop motions, secondary structure changes and hinged domain movements	Microseconds-milliseconds	kex ≈ 200–6,000 s−1 kex ≈ Δω	37,82,97
R1ρ rotating frame relaxation dispersion (RF RD)	Attenuation of chemical exchange broadening by applying refocusing pulses: reduce relaxation R1ρ sensitive to motions	Fast-intermediate conformational exchange such as motions of loops, side chains and secondary structure elements	Microseconds -milliseconds	kex ≈ 10,000–50,000 s−1 kex ≥ Δω	5
Paramagnetic Relaxation Enhancement (PRE)	Increased rate of relaxation due to magnetic dipolar interaction between a nucleus and an unpaired electron of a spin label	Lowly populated states of macromolecules and their complexes, non-specific interactions, ligand binding sites, fast dynamic processes	Microseconds	kex ≈ 100,000 s−1 kex ≫ Δ ω	15
R1, R2 and heteronuclear NOE relaxation (nuclear spin relaxation)	Analysis of longitudinal and transverse relaxation rates and the associated heteronuclear NOEs, sensitive to protein motions	Fast conformational exchange such as bond vibration and libration, side chain rotamer interconversion, random coil, loop motions, and backbone torsion angle rotation	Picoseconds - nanoseconds		16,98
Residual Dipolar Coupling (RDC)	Combined analysis of dipolar coupling constants measured in an array of experimental conditions	Structural states of a protein in solution, orientation of bond vectors, fast conformational exchange	Picoseconds-milliseconds		17,99
Chemical Shift Covariance (CHESCA) or Projection (CHESPA) Analysis	Analysis of residue networks that show correlated changes in chemical shifts due to perturbations in the system (mutations, ligand binding, etc.)	Allosteric networks, ligand binding sites			43,44,49–51

^aThe exchange rate (k_ex) is proportional to the chemical shift difference (in Hertz) between two exchanging states (Δω).