NMR screening

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1D 1H NMR Screening

Initial screening of NMR samples can be done with 1D 1H NMR on labeled or unlabeled protein samples in aqueous buffer with 5-10% D2O.

It is helpful to have an estimation of the protein concentration obtained from a method such as UV absorption and also to add 50 uM DSS as an internal standard for both referencing (to 0 ppm) and for estimating the protein concentration.

The criteria for judging the NMR spectrum are:

  1. good signal to noise as compared to the DSS peak
  2. chemical shifts dispersion upfield of 0 ppm is good, since it indicates methyl protons that are in a folded core and nearby to methyl groups (not essential if the protein does not have aromatic residues)
  3. chemical shifts dispersion in the amide region is good (will be much less if protein is predicted to be mostly helical vs. mosty beta strand.
  4. peaks should not be too broad, allowing resolved peaks to be observed
  5. uniform intensity of peaks in the amide regions is ideal.

At Rutger's University, samples are screened at 20 degrees using 1.7-mm NMR tubes that are loaded into a Bruker B-ACS 60 samples handler and run on a Bruker TCI 1.7 MicroCryoprobe.

Samples with promising 1D spectra should be 15N labeled so that the [15N-1H] HSQC can be recorded.

2D [15N, 1H] HSQC for peak dispersion and peak yield assessment

The following peaks are alway visible in [15N, 1H] HSQC:

  • 1 peak for each backbone amide except that of the first residue
  • 1 peak for each Trp side chain HE1/NE1 group, usually located in the most downfield region.
  • 4 peaks for each Asn and Gln side chain - these are ND2/HD21, ND2/HD22 of -CONH2 and -CONHD groups. -CONHD peaks appear due to the exchange with D2O and have distinct chemical shifts due to deuterium isotope effects. Usually CONHD peaks are much weaker and located above the CONH2 peaks.

The following peaks are also sometimes observed in [15N, 1H] HSQC:

  • NE/HE of Arg (a few are normally visible in every protein, 15N shift ~90 ppm)
  • NH1/HH11, NH1/HH12, NH2/HH21, NH2/HH22 of Arg (rarely visible, usually broad, 15N shift ~70 ppm)
  • ND1/HD1 and/or HE2/HE2 of His (rarely visible, 15N shifts vary greatly, can be mistaken for Trp)