Wikiomics:Nuclear Magnetic Resonance
Biological Nuclear Magnetic Resonance or NMR is a technique which allows the determination of full or partial 3-dimensional structures of macromolecules. As opposed to Wikiomics:X-ray crystallography, NMR does not require the molecules to be arranged periodically in a crystal and therefore, mixture of molecules in different conformations can be studied dynamically.
Basics of NMR
NMR can detect transition of nuclear spins. The most useful nuclei are those with nuclear spin 1/2. Many elements have such isotopes. Some are naturally abundant (1H) but most are not (13C, 15N)
In order to perform most modern experiments, it is necessary to produce, isotopically enriched proteins. This is achieved by heterologous expression in NMR:Minimal Media. In this case, the nitrogen and carbon sources are controlled and the desired isotopic enrichment can be achieved. Selective labeling of proteins is more complicated.
Limitations of NMR
The size of the protein is a big problem with NMR. As a rule of thumb, 20kDa is the limit for traditional NMR. For proteins above that size, it is necessary to work with Deuterated samples NMR:Deuteration
NMR suffers from low sensitivity so the samples have to be sufficiently concentrated. Usually 500ul of 1mM sample is optimal for collecting all the needed experiments, however, with the current developments in Instrumentation (See crycrobes and nanoprobes) it is possible to work with a few mg of protein
For the compete structure determination, 3 different sample should be prepared
- Unlabeled Sample
- U-15N labeled sample
- U-13C15N labeled sample