Sample Requirements
For structure determination you will need a roughly 1 mM solution. Standard 5 mm tubes require 500 μL, while 1.7 mm capillary tubes require 40 μL. The purity needs to be > 95%, as extra NMR signals complicate the analysis dramatically. As the molecular weight of the protein increases the number of NOEs measured, and their tendency to overlap, increases. To improve resolution, spectra with additional dimensions using nuclei such as 13C or 15N can be recorded, but this requires producing isotopically labelled protein. The table below lists the isotopic labelling required for different sized proteins. Note that these limits are for favourable cases.
| # Residues | ~ MW (kDa) | Isotopes |
|---|---|---|
| 1-50 | <5 | none |
| 50-100 | 5-10 | 15N |
| 100-250 | 10-25 | 13C and 15N |
| 250-400 | 25-40 | 2H, 13C and 15N |
Site specific labelling can be used for proteins greater than 400 residues, but will only give information on the labeled sites. Isotopic labelling requires expressing the protein in minimal media supplemented with isotopically labelled material; typically ammonium sulfate or chloride for 15N, glucose for 13C, or D2O for 2H.
Proteins and peptides are typically dissolved in aqueous solution for NMR analysis. To reduce the exchange of amide protons with the bulk water the pH should be slightly acidic, although up to pH 7.4 can be used. The buffer should not add extra signals to the NMR spectra, so phosphate buffer is the best choice. If the sample is isotopically labelled then this is not as much of an issue, since the non-labelled buffer molecules will not be detected.
Data Collection
NOESY spectra provide the NOEs to determine the three-dimensional structure. A 2D NOESY spectrum can be recorded in a few hours, however, for proteins larger than roughly 50 residues the peaks in a 2D spectrum will be too overlapped and so 3D spectra need to be recorded. A 3D NOESY spectrum can take from several hours up to 2 days to record.
In addition to the NOESY spectra, assignment spectra will need to be recorded. These spectra allow the atomic interactions responsible for the NOEs to be identified. For peptides and small proteins, 2D DQF-COSY and TOCSY spectra will be sufficient. For proteins labelled with 15N only, 3D versions of these experiments are used. Labelling with 13C enables the use of triple resonance experiments where the interactions of 1H, 13C and 15N nuclei are rigorously selected. Triple resonance experiments are typically used in pairs, where one experiment shows only intra-residue correlations and the other shows intra- and inter-residue correlations.
For a 13C labelled protein complete data collection could take up to two weeks. Innovations such as NUS1 and BEST2 are reported to reduce the time taken to roughly 24 hours, but the Skaggs NMR Facility has limited experience with these at present.
Assignment
Before the NOEs measured in the NOESY spectra can be used to calculate a structure, the protons responsible for them must be identified. This necessitates the assignment of every proton in the molecule. Triple resonance experiments make this fairly simple by transferring magnetisation via well known scalar coupling, thereby limiting the possibilities. However, analysis of such spectra still requires comparing at least two three-dimensional cubes of data. Free software for doing this is available3,4, but requires some training. A new user might take two weeks to learn how to navigate the data and completely assign a protein. For smaller proteins without 13C labelling, assignment relies on comparison of TOCSY and NOESY spectra and the assumption that the additional peaks in the NOESY spectra are due to interactions between atoms in sequential residues.
Assignment of the protein identifies secondary structure elements of the protein, even without using NOEs, because α-helices and β-strands induce characteristic movement of the chemical shifts. In some cases this may be sufficient and a full structure determination may not be required.
Structure calculation
To calculate the structure an all-atom, three-dimensional model of the protein is manipulated to sample as many configurations as possible while distance restraints from the NOEs are applied. This means that the planar atom connectivity must be known. Free structure calculation software is available5,6, but the most effective package7 is not free. Much of the time taken to calculate a structure is taken up resolving restraint violations by correcting NOE assignments, editing peak lists, and adjusting peak volumes.
Running structure calculations inevitably requires that the user is comfortable with, or at least willing to learn, linux, command line tools, and some scripting languages. Structure calculations are likely to take at least a few weeks to produce acceptable results.
References
1. Mobli M, Maciejewski MW, Schuyler AD, Stern AS, and Hoch JC.
Sparse sampling methods in multidimensional NMR.
Phys Chem Chem Phys. 2012;14(31):10835-10843
2. Lescop E, Schanda P, and Brutscher B.
A set of BEST triple-resonance experiments for time-optimized protein resonance assignment.
J Magn Reson. 2007;187(1):163-169
3. Lee W, Tonelli M, and Markley JL
NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy.
Bioinformatics. 2015 Apr 15;31(8):1325-7
4. Skinner SP, Fogh, RH, Boucher W, Ragan TJ, Mureddu LG, Vuister GW
CcpNmr AnalysisAssign: a flexible platform for integrated NMR analysis
J Biomol NMR. 2016 Oct 1;66(2):111-12
5. C.D. Schwieters, J.J. Kuszewski, N. Tjandra and G.M. Clore
The Xplor-NIH NMR Molecular Structure Determination Package
J. Magn. Res. 2003;160:66-74
6. D.A. Case, T.E. Cheatham, III, T. Darden, H. Gohlke, R. Luo, K.M. Merz, Jr., A. Onufriev, C. Simmerling, B. Wang and R. Woods.
The Amber biomolecular simulation programs.
J Computat Chem. 2005;26:1668-1688
7. Güntert, P. and Buchner, L.
Combined automated NOE assignment and structure calculation with CYANA
J Biomol NMR. 2015;62:453-471
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