How to make your samples for NMR study?
Usually the proteins are expressed with E.Coli in either rich medium or isotopically enriched medium (13C glucose and 15N NH4Cl) and purified by FPLC or HPLC. It will be the best if your lab is equipped to do so. Otherwise, you can use service from the Recombinant Protein Production Core (rPPC) at Northwestern to make the unlabeled and C13 and/or N15 labeled proteins. Their website is
The NMR sample is normally buffered at pH below 7. The volume is 500 uL. It should contain 10% D2O for locking purpose.
What would be the first NMR experiments to run and what biomolecular information can be obtained from them?
Although solution NMR can provide extremely valuable information on the three-dimensional structure of proteins and protein complexes in solution, the molecular motions at various timescales using NMR relaxation, and the ligand binding site etc., the easiest way to use it for non spectroscopist is to evaluate the protein folding, aggregation, and dynamics. Once a protein is confirmed to be well folded by NMR, one can try to crystalize it because the x-ray crystallography is still the quickest way to obtain three dimensional structures for proteins. If you have protein crystal, you can use the service at Center for Structural Biology (CSB) to get the x-ray structure much quicker than using NMR. (http://www.csb.northwestern.edu/)
Figure 1 (AH Kwan etc., FEBS Journal, 2010) shows the 1D protein and 2D 15N-HSQC spectra for six different proteins. You can see the contrasting characters of folded proteins vs unfolded ones, alfa-helical vs beta-strand, etc. For details, please see the figure captains.
Figure 2 (same reference) shows NMR spectra features for protein, oligonucleotide, and polysaccharide. The protein has most dispersive frequency distribution and the polysaccharide is the poorest. The oligonucleotide falls in between.
Figure 1: 1D 1H‐ and 15N‐Figure 1: 1D 1H- and 15N-HSQC spectra of (A) AHSP, a 10 kDa all‐α‐helical protein; (B) EASΔ15, a 7 kDa predominantly β‐sheet protein; (C) PRD‐C6, a disordered 6 kDa polypeptide; (D) EAS, an 8 kDa predominantly β‐sheet protein that contains a 19 residue disordered region; (E) PRD‐Xb, a 12 kDa protein segment that exists in a molten globule state; and (F) YPM, a 14 kDa protein for which approximately 25% of the residues are involved in μs–ms dynamics.
Figure 2: (A) 1D 1H‐NMR spectrum, (B) 15N‐HSQC spectrum and (C) 13C‐HSQC spectrum of CtBP‐THAP, a 10.6 kDa protein. Sidechain amide groups from Asn and Gln residues are indicated by dotted lines. All three spectra were recorded on a 1mM sample in 20 mM sodium phosphate (pH 6.5) containing 100 mm NaCl and 1 mM dithiothreitol at 298 K on a Bruker 600 MHz spectrometer (Bruker, Karlsruhen, Germany) equipped with a cryoprobe. The spectrum in (A) was recorded over 30 s, whereas the 13C‐ and 15N‐HSQC spectra were recorded over 5 min. (D) 1D 1H‐NMR spectrum of a 19 bp (11.7 kDa) double‐stranded DNA oligonucleotide. (E) 1D 1H‐NMR spectrum of a polysaccharide. Note the poor signal dispersion compared to the protein spectrum.
How to do sequence-specific backbone resonance assignments for a double-labeled protein?
For any double-labeled protein, a whole suite of triple resonance experiments for backbone resonance assignments are collected. After being processed with NMRPIPE (the most popular bionmr processing packaged), the data then can be analyzed and chemical shifts be assigned. The following link provide an excellent explanation on the principle of this procedure: http://www.protein-nmr.org.uk/solution-nmr/assignment-theory/triple-resonance-backbone-assignment/
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