Initial steps. The first step is to be sure that you can synthesize and purify a sufficient quantity of protein (or other biomacromolecule) and that it can be solubilized. Many different solvent systems are acceptable, but you should be aware that NMR signal-to-noise is degraded by high salt concentrations and that NMR lines become broad if the solvent viscosity is high. The NMR instrumentation at NMRFAM collects data only on liquid samples. Owing to the low natural abundance of 13C and 15N, observation of these nuclei usually requires that they be enriched. In some limited cases, natural abundance levels of the isotopes are sufficient.
Testing. NMR studies of biomolecules usually take at least one day; a week or two of data collection may be required for a structure determination of a protein or nucleic acid. The first criterion for success is that the sample must remain stable over this time period. The spectrometers have variable temperature capabilities, and the temperature of the sample can be adjusted between 0oC and 50oC to optimize the stability of your system. Raising the temperature above ambient typically gives higher quality spectra (because molecules tumble more rapidly) while lowering it may lead to lowered resolution. A second criterion for success is the spectral quality achieved with the sample under the solution conditions used, such as pH, buffer, additives, temperature.
Although it may be possible to determine the stability and spectral quality of a sample without labeling, more definitive results can be obtained with a sample labeled uniformly with 15N. A solution of a few milligrams of protein is prepared, and an initial test spectrum (a HSQC spectrum that takes 0.5-24 hr) is collected. This spectrum generally is diagnostic of further success. It may be necessary to adjust the sample conditions, such as pH, salt, buffers, to improve the quality of the spectrum. Sometimes, a redesigned molecule (protein or nucleic acid with altered sequence) will yield better quality spectra. Once a high quality spectrum is obtained, the sample is retested a week or other suitable time later to establish the stability of the sample. Obviously, the length of time that the sample must remain stable must be long enough to collect the data. Therefore, if you want to determine the structure of a large biomacromolecule, it must remain stable for a week or longer.
Once it is determined that the protein is suitable for 3D structure determination, a 13C and 15N labeled sample (double labeled) is prepared for data collection. If you have a large (>20-30 kDa) biomacromolecule or a large (>20-30 kDa) biomacromolecular complex, you may need to also replace carbon bound hydrogens with deuterium in addition to 13C and 15N labeling (triple labeling).
Special labeling schemes (selective, 13C, 15N, or both) may be used for assignments, dynamics, ligand binding, pH titration, and other studies. If you contact the facility, our experienced staff can help you determine the best labeling scheme for your experiment.