What is NMR?
Nuclear Magnetic Resonance (NMR) spectroscopy takes advantage of the natural magnetism present in certain atomic nuclei. The nuclei most often studied in biological applications of NMR are: 1H, 13C, 15N, and 31P. All of these nuclei are stable (non-radioactive) isotopes. When a molecule is placed in a magnetic field, these nuclei act like tiny little bar magnets and align like iron filings. The nuclear bar magnets can align either in the same direction (up) or in the opposite direction (down) as the external magnetic field; these two alignments have different energies. An analogy of this energy difference can be physically experienced by holding the north poles of two magnets together versus holding the north and south poles together. In the first case, they repel and in the latter they attract. It is the difference in energy between these two alignments of the nuclei in the magnetic field that is the fundamental quantity measured by NMR. The great strength of the technique comes from the fact that the energy difference between the up and down states (a.k.a. the chemical shift) is exquisitely sensitive to the molecular environment of the nucleus. Virtually every nucleus in a molecule experiences a different environment and thus has a chemical shift different from that of every other nucleus. For example, the chemical shift of a C atom bonded to another C and three H atoms will be different from that of a C bonded to two C and two H atoms. This means the structure of a molecule is encoded in the chemical shifts of the nuclei. By using rather sophisticated experiments, the chemical shift of every nucleus in the molecule and its physical location in the molecule can be determined.
Just as the nuclear bar magnets in a molecule interact with the external magnetic field, they also interact with each other. The strength of this interaction depends on the distance between the nuclei. This feature makes it possible to use NMR to determine distances between nuclei that are closer than about 5Å. Thus NMR can be used to determine the folded conformation of a protein or nucleic acid or to discover how a drug binds to its receptor molecule. Three-dimensional structures of molecules are determined by combining information on chemical shifts assigned to particular atoms with internuclear distances estimated from NMR experiments. Specialized NMR experiments can be used to determine how rapidly the molecule or parts of the molecule are moving in solution. The time scale of motions over which NMR is sensitive covers an enormous range: 10-11s to >1s. In the picosecond to nanosecond time range, effects of librations, vibrations, and overall molecular tumbling can be quantified. The microsecond to the second time frame includes motions involved in conformational changes, ligand binding, and catalysis.
Diffusion measurements, kinetics, metabolism, oil well logging, imaging, fluid flow, analysis of foodstuffs, analysis of zeolite catalysts, clinical quantification of serum cholesterol, combinatorial chemistry, and quantum computing are among the far-ranging applications of NMR. All of this versatility comes at a cost. NMR spectroscopy is an insensitive spectroscopic technique. The problem arises from the very small energy differences between the up and down states of alignment. The experimental problem is the detection of the weak absorption of photons that excite the excess population in the ground state. Due to the lack of sensitivity, liquid state NMR requires micromoles of compound at relatively high concentration (1 mM) and detection by very expensive equipment. NMRFAM supplies the equipment and expertise necessary to do these types of experiments.
NMR seems very complicated. Do I need a complete understanding of NMR in order to use the facility and obtain data?
No. The theory behind NMR is very complicated, but you do not need to have an in-depth understanding of it in order to collect data to answer a particular biochemical question or obtain a structure. This is analogous to an MRI scan in a hospital the doctor does not need to understand the theory of the instrument in order to do the scan and interpret the data. The NMRFAM staff includes experts in multiple areas of NMR spectroscopy who can train you to run the samples and analyze the data. The staff is also available to answer questions. Eight hours of training are provided for new users free of charge. If additional training is needed, the fee is $50/hour. The training occurs on-site, although questions can be answered via phone or e-mail. If you need training or have questions, please contact the facility Site Administrator, David Aceti at firstname.lastname@example.org.
If you have a small molecule and do not want to perform the experiment yourself, you can submit a sample to NMRFAM, and we will collect standard spectra for you. Service
Is there a way to find out if my biomacromolecule is amenable to NMR analysis?
Yes, several questions that should be addressed first (below). Our staff can help you determine the best experimental approach for your particular question.:
1 – What problem do you want to study? NMR is very good at some and poor at others.
2 – What is the molecular mass of your biomacromolecule and does it dimerize or oligimerize? If it is ~20 kDa or more, there may be limits on what can be studied.
3 – How much sample can you produce? Is it soluble at the concentration needed by NMR, and can you incorporate stable isotopes.
Is there limit to how large my biomacromolecule can be?
It generally depends on the type of information that is desired. Structural studies are the most limited case. The molecular mass of most biomacromolecules under investigation by NMR are less than ~20-25 kDa with the majority < 20 kDa. If you want to investigate the binding of ligands or determine the binding surface in a protein-protein interaction, molecules with much higher masses can be studied.
New techniques and stable isotope labeling methods have greatly increased the maximum molecular mass of biomacromolecules available for study by NMR. The structures of several b-barrel membrane spanning proteins inserted in a micelle have been determined. These systems had an aggregate molecular mass of ~50-60 kDa. Generally, structure determinations of proteins have been limited to molecules with less than ~400 amino acid residues, however, the global fold for a 82 kDa single chain protein in solution has been reported in Tugarinov et al., PNAS 102(3)622-627 (2005), and there are some reported studies of protein complexes with molecular masses between 200 and 800 kDa. For reviews, see Tugarinov, et al., Ann Rev Biochem 73:107-146 (2004) and Riek et al., TIBS 25(10):462-468 (2000).