NMR is a non-destructive technique and simple 1D 1H-experiments to ascertain aggregation state and purity can often be run in a matter of minutes or hours if sufficient sample is available and the molecular weight of the species in solution is below 20 kDa.
Typically, these experiments can determine if the sample is pure, bound to a ligand, aggregated, unfolded, or acting as a molten globule with defined secondary structure but ill-defined tertiary structure. The use of these experiments to determine the behaviour of the molecule of interest in solution depend, among other things, on a careful examination of line width and signal dispersion in the proton (1H) spectrum.
The sample requirements for these experiments are ca. 300 microliters of protein with a concentration above approximately 25μM.
In some cases, the analysis standard 2D or 3D 1H-experiments usng unlabelled samples can be be employed to assign the signals in an NMR spectrum and allow a structure to be calculated. However, typically modern experiments require isotopically labelled samples…
Luckily, the belief that labelling of proteins is expensive no longer holds. One gramme of 15N ammonium chloride, which is enough for 1 litre of M9 minimal medium now only costs ca. £20. The 3 grammes of uniformly 13C-labelled glucose required for 1 litre of the minimal medium required for the complete assignment of a protein of 200 amino acids only costs £200.
Labelled samples allow a wide variety of experiments to be carried out. For well-behaved proteins of up to 80 to 100 amino acids it is possible to achieve a structure with just 15N-labelling. For well-behaved proteins up to 200 amino acids in length it is possible to calculate a structure or achieve assignments suitable for analysis of dynamics or ligand binding using samples which are doubly labelled in 15N and 13C.
For proteins having more than 200 amino acids more sophisticated experiments and sample preparation including perdeuteration and site-specific, i.e. selective, labelling are required. Combining these labelling approaches with state-of-the-art triple-resonance NMR experiments, useful data can be acquired on very large proteins and proteins bound to membrane mimetics of up to ca. 100kDa in molecular weight.
The NMR facility here in Leeds houses a brand new 950 Bruker Ascend Aeon™ NMR spectrometer and two Oxford NMR magnets operating at proton resonance frequencies of 750 MHz and 600 MHz, respectively. All three magnets are equipped with Bruker Avance III HD consoles and the latest state-of-the-art cryoprobe technology featuring Bruker TXO-, TCI-, and QCI-P-cryoprobes, respectively. NMR experiments can be carried out using any of our three NMR spectrometers. The choice of instrument used in an experiment very much depends on the size and type of system being examined and/or the NMR experiment being used. In some cases, spectra need to be recoreded at multiple magnetic fields to enhance the quality of the NMR data.
For a short series of experiments the NMR facility can provide a member of staff to acquire and process spectra. For long-term projects, the researchers will be trained to use the instruments and process data themselves. In many cases, the user will be expected to enter into a collaboration with the NMR facility. Some of our instruments are heavily used, and as experiments can last for several days or weeks we have a booking meeting to organise the booking of the magnets for the following week. However, at the start of a project, we would suggest you contact the facility staff members so they can assist in designing your project. NMR is a sophisticated analytical technique and high quality advice at the start of a project is essential for its success.
In order to cover the running and maintenance costs of the instruments, researchers are charged for using the NMR spectrometers. Current charging rates can be provided upon request. The NMR facility also has a wealth of experience in the labelling and stabilisation of biomolecules in solution, which we are happy to provided to prospective users. The facility also has a stock of chemicals for the growth of proteins in minimal media, deuterated solvents, buffer components, amino acids, sugars, and NMR tubes.