Nuclear magnetic resonance is a phenomenon that occurs because some atomic nuclei have magnetic properties. In NMR, these properties are utilized to obtain chemical information. Subatomic particles can be thought of as spinning on their axes, and in many atoms these spins balance each other such that the nucleus itself has no overall spin. However, these spins do not balance out in 1H, 13C, 15N, 19F and 31P. Such nuclei can have one of two possible half spins both of which have the same energy.
Nuclei with half spin behave like a magnet. When placed in constant magnetic field, they tend to align themselves with the field. When placed in a magnetic field, the energy level splits as in one orientation the nucleus aligns itself with the magnetic field while it cannot align itself if it is in another orientation. Where such energy separations exist, nuclei can be induced to jump from the lower-energy magnetic spin state to the less favorable higher-energy state when exposed to radio waves of a certain frequency. This absorption is called resonance because the frequency of the radio waves coincides with the frequency at which the nucleus spins. When the nuclei flip back to their original orientations, they emit radio waves that can be measured. Protons 1H give the strongest signals, and this is the basis of protein structural analysis by NMR spectroscopy.
To create this a constant magnetic field, NMR spectrometers contain superconducting magnets. Superconducting magnets have no resistance and no current loss. They require cooling to almost absolute zero. In brief, NMR is very expensive.
The energy input to make the nuclei resonate is produced by radio frequency (RF) pulse. Different effects are measured based on the length of the pulses and delay between the pulses. Thus an NMR spectra can generate a large variety of spectra. Depending on the frequency of the RF pulse, different nuclei can be detected. After the pulse, the nuclei return to their ground energy state. The nuclei precess back to their start position and this precessing induces a current which is detected by a coil in the NMR spectrometer. This is act of returning back to ground state is call Free Induction Decay (FID).
A free induction decay (FID) is the observable NMR signal generated by non-equilibrium nuclear spin magnetisation precessing about the magnetic field (conventionally along z). This non-equilibrium magnetisation is generally created by applying a pulse of resonant radio-frequency close to the Larmor frequency of the nuclear spins. [5]
AN NMR spectrum is a superposition of signals, one FID per signal. Fourier transforms are used to transform FID from time domain to frequency domain.
Step in NMR spectroscopy
- NMR experiments
- data collection
- spectrum assignment
- structure calculation
NMR spectroscopy is used to determine the structures of proteins in solution, and this requires proteins to be both highly soluble and stable.
One dimensional NMR experiments can detect chemical shifts and other shielding effects such as spin-spin coupling. One dimensional NMR is generally insufficient to characterize complex molecules like proteins. Therefore, instead of using a single radio pulse, a sequence of pulses are used separated by different time intervals which give a two dimensional NMR spectrum with additional peaks indicating pairs of interacting nuclei. Three types of interactions can be measured by using different pulse sequences.
COSY - correlation spectroscopy
It detects sets of protons interacting through bonds, i.e. protons linked to adjacent bonded pairs of C and N atoms allowing us to trace a network of protons linked to bonded atoms.
TOCSY - total correlation spectroscopy
It detects groups of protons interacting through a coupled network, not just those joined to adjacent bonded pairs of C or N atoms. TOCSY can often identify all the protons associated with a particular amino acid, but cannot spread to adjacent residues because there are no protons in the carboxyl portion of the peptide bond.
NOESY - nuclear overhauser effect spectroscopy
It takes advantage of the nuclear overhauser effect i.e. signals produced by magnetic interactions between nuclei that are close together in space but not associated by bonds. This is useful for determining protein structures because interactions can be identified between protons that are widely separated along the polypeptide backbone but close together in space due to the way in which the protein folds.
When these effects are taken into account, the result of NMR analysis is a set of distance constraints, which are estimated distances between particular pairs of atoms (either bonded or unbonded). If enough distance constraints are calculated, the number of protein structures that fit the data becomes finite. Thus NMR analysis produces 10-50 models instead of a unique structure. Good NMR resonance depends on the protein molecule tumbling rapidly in the solvent, which limits the size of proteins that can be analyzed to those with fewer than 300 residues.
Distance geometry, simulated annealing, and torsion angle dynamics are used to calculate structures.
X-ray crystallography tends to produce more accurate models than NMR although where both methods have been applied to the same protein there appears to be excellent agreement in the structures. This is probably because protein crystals have large water contents and thus exist in similar state to dissolved proteins. An important advantage of NMR is that it is possible to measure the dynamics of each residue with this method and it can therefore distinguish between regions of the protein that vibrate and those that are disordered. NMR also provides positions of many hydrogen atoms, which is not possible with x-ray crystallography.
Advantages
- no chemical modification necessary
- protein in solution: no crystal packing artefacts, allows direct binding experiments, hydrodynamic and folding studies
- assignment of labile regions possible: no gaps in structure
Disadvantages
- protein in solution: protein has to be soluble
- insensitive method: requires high concentrations of proteins
- overlap: direct determination of 3D structures for small
- proteins only (150-200 residues)
NMR spectroscopy is used routinely in high-throughput screens to determine protein:ligand interactions.[2][3] NMR is also a key tool in mechanistic enzymology and in studies of protein folding and stability.
Source
[1] Principles of Proteomics by R. M. Twyman
[2] Hajduk et al., 1999
[3] Shuker et al., 1996
[4] Structural Bioinformatics by Bourne & Weissig
[5] Wikipedia.org