What information can you get from NMR?

Localising interactions and conformational changes

• NMR chemical shift is exquisitely sensitive to environment, and site-specific binding can be mapped to sequences and structures by measuring chemical shift perturbations (CSP).

• Identification of the location of binding sites can be determined for both strong and weak interactions.

• Interactions can be quantified and assessed for specificity even in the absence of assignments.

Analyse protein mutations

• NMR provides rapid insights into whether mutation effects are local or long-range, and so whether large-scale conformational changes are occurring.

Diagnostics and sample quality control

• An NMR spectrum provides a ‘fingerprint’ of a protein that depends on the chemistry, conformation, and environment of the sample. Overlaying two spectra indicates whether or not the samples are identical.

Investigate IDPs and intrinsically disordered regions (IDRs)

• Disordered proteins can be characterised and residual structure quantified. Flexible regions within even very large systems can be detected (e.g., ribosome) and characterised.

• Detecting residual structures and transient interactions in phase-separating proteins.

Small molecule binding, including pooled screens

Protein-detected experiments:

• site-specific binding information

• very good at detecting weak interactions (e.g., with small molecule fragments)

• pooled molecule screening possible followed by deconvolution

• normally requires 15N-labelled samples at 10's µM, and protein chemical shift assignments

Ligand-detected experiments:

• 1D-based 1H spectra, so sensitive and fast

• Can use much less protein (low µM) and protein is unlabelled

• A very wide range of Kd's can be determined

• well suited for pooled library screening and easy to deconvolute

• possible approaches include STD-NMR, WaterLOGSY, transferred NOE, relaxation editing, diffusion editing.

 

Identifying and/or quantifying dynamics on any timescale

NMR chemical shifts and linewidths are very sensitive to local motion and can be used to study directly functionally important motion that is only inferred from XRC or cryoEM data:

Type of Motion

Time scale

NMR experiment(s) for detection

local, uncoupled motion ('intrinsic' disorder)

ps-ns

15N T1 & T2; 1H-15N heteronuclear NOEs

loop motions in active sites or regulatory regions

µs-ms

T1ρ, CPMG (relaxation dispersion), or CEST

cooperative motion of larger regions of structure; folding/unfolding events

seconds to hours

ZZ exchange, H/D exchange

 

'Real-time' NMR

Any event with a spectroscopic signature and occurring on a timescale of minutes to hours can be monitored in the spectrometer to extract kinetics information.

Thermodynamics 

• NMR data can be recorded under a range of temperatures (5-60˚C), providing information on thermodynamics for structures and interactions.

'In cell' NMR

• Labelled proteins or other molecules can be injected into cells, or in some cases, selectively produced labelled in cells, to allow for studies of structure, dynamics, etc., in the cellular context.

Other information from NMR:

• Protonation states, pKa's, etc.

• Quick determination of effects from solvent (pH, salt, etc.) or temperature (~5˚C-60˚C) 

• Determine secondary structure propensities of fully or partially folded regions (chemical shifts)

• Solvent exposure of amino acids in a protein (e.g., using H/D exchange)

• Local structural information (e.g., individual dihedral angles

• Diffusion: both rotational diffusion (τc) and translational diffusion coefficient (D)