Below is a Google-cached version of Protein NMR - A Practical Guide - Solution NMR Spectra page from www.protein-nmr.org.uk crashed on 11/6/11. You may want to check first if the site has been restored.
Spectrum Descriptions
This page contains a list of the solution NMR experiments most commonly used in protein NMR assignment and structure calculation. For each experiment there is an illustration showing which atoms are observed (pink) and through which atoms magnetisation flows (light blue). An example spectrum shows what it should look like and particular features and considerations are highlighted. The descriptions here are very simplistic and for a full description, including pulse sequences and product operator treatment, the reader should consult the original papers or a book such as
Protein NMR Spectroscopy - Principles and Practice by J. Cavanagh, W. Fairbrother, A.G. Palmer III and N.J. Skleton (Academic Press) (
Link to Publisher).
1H-15N-HSQC (2D)
References:
see
Protein NMR Spectroscopy - Principles and Practice by J. Cavanagh, W. Fairbrother, A.G. Palmer III and N.J. Skleton (Academic Press). (
Link to Publisher)
min. labelling: 15N
Magnetization is transferred from hydrogen to attached 15N nuclei via the J-coupling. The chemical shift is evolved on the nitrogen and the magnetisation is then transferred back to the hydrogen for detection.
This is the most standard experiment and shows all H-N correlations. Mainly these are the backbone amide groups, but Trp side-chain Nε-Hε groups and Asn/Gln side-chain Nδ-Hδ2/Nε-Hε2 groups are also visible.
The Arg Nε-Hε peaks are in principle also visible, but because the Nε chemical shift is outside the region usually recorded, the peaks are folded/aliased (this essentially means that they appear as negative peaks and the Nε chemical shift has to be specially calculated). If working at low pH the Arg Nη-Hη and Lys Nζ-Hζ groups can also be visible, but are also folded/aliased.
The spectrum is rather like a fingerprint and is usually the first heteronuclear experiment performed on proteins. From it you can assess whether other experiments are likely to work and for instance, whether it is worth carbon labelling the protein before spending the time and money on it. Or if your protein is reasonably large you might be able to judge whether deuteration might be necessary.
Top
HNCO (3D)
References:
L.E. Kay, M. Ikura, R. Tschudin and A. Bax (1990)
J. Magn. Reson. 89 496-514. (
Link to Article)
S. Grzesiek and A. Bax (1992)
J. Magn. Reson. 96 432-440. (
Link to Article)
D.R. Muhandiram and L.E. Kay (1994)
J. Magn. Reson., Ser. B 103 203-216. (
Link to Article)
min. labelling: 15N, 13C
Magnetisation is passed from 1H to 15N and then selectively to the carbonyl 13C via the 15NH-13CO J-coupling. Magnetisation is then passed back via 15N to 1H for detection. The chemical shift is evolved on all three nuclei resulting in a three-dimensional spectrum.
This is the most sensitive triple-resonance experiment. In addition to the backbone CO-N-HN correlations, Asn and Gln side-chain correlations are also visible.
It is mainly used to obtain CO chemical shifts which can be used in a program like
TALOS to help predict secondary structure.
The HNCO can also be useful for backbone assignment in conjunction with the HN(CA)CO, if the CBCANNH and CBCA(CO)NNH spectra are of bad quality.
Top
HN(CA)CO (3D)
Reference:
R.T. Clubb, V. Thanabal and G. Wagner (1992)
J. Magn. Reson. 97 213-217. (
Link to Article)
min. labelling: 15N, 13C
The Magnetisation is transferred from 1H to 15N and then via the N-Cα J-coupling to the 13Cα. From there it is transferred to the 13CO via the 13Cα-13CO J-coupling. For detection the magnetisation is transferred back the same way: from 13CO to 13Cα, 15N and finally 1H. The chemical shift is only evolved on 1H, 15N and 13CO and not on the 13Cα. The result is a three-dimensional spectrum. Because the amide nitrogen is coupled both to the Cα of its own residue and that of the preceding residue, both these transfers occur and transfer to both 13CO nuclei occurs. Thus for each NH group, two carbonyl groups are observed in the spectrum. But because the coupling between Ni and Cαi is stronger than that between Ni and Cαi-1, the Hi-Ni-COi peak generally ends up being more intense than the Hi-Ni-COi-1 peak.
This experiment can be useful for backbone assignment when used in conjunction with the HNCA, HN(CO)CA and HNCO if the CBCANNH and CBCA(CO)NNH spectra are of bad quality.
An overlay of the HNCO and HN(CA)CO spectra makes it very easy to distinguish between COi and COi-1 for each NH group.
Top
HNCA (3D)
References:
L.E. Kay, M. Ikura, R. Tschudin and A. Bax (1990)
J. Magn. Reson. 89 496-514. (
Link to Article)
B.T. Farmer II, R.A. Venters, L.D. Spicer, M.G. Wittekind and L. Müller (1992)
J. Biomol. NMR 2 195-202. (
Link to Article)
S. Grzesiek and A. Bax (1992)
J. Magn. Reson. 96 432-440. (
Link to Article)
min. labelling: 15N, 13C
Here the magnetisation is passed from 1H to 15N and then via the N-Cα J-coupling to the 13Cα and then back again to 15N and 1H hydrogen for detection. The chemical shift is evolved for 1HN as well as the 15NH and 13Cα, resulting in a 3-dimensional spectrum. Since the amide nitrogen is coupled both to the Cα of its own residue and that of the preceding residue, both these transfers occur and peaks for both Cαs are visible in the spectrum. However, the coupling to the directly bonded Cα is stronger and thus these peaks will appear with greater intensity in the spectra.
This experiment can be useful for backbone assignment when used in conjunction with the HN(CO)CA, HNCO and HN(CA)CO if the CBCANNH and CBCA(CO)NNH spectra are of bad quality.
By overlaying the HN(CO)CA spectrum with the HNCA, it becomes even easier to identify and distinguish between all Cαi and Cαi-1 peaks.
Top
HN(CO)CA (3D)
References:
A. Bax and M. Ikura (1991)
J. Biomol. NMR 1 99-104. (
Link to Article)
S. Grzesiek and A. Bax (1992)
J. Magn. Reson. 96 432-440. (
Link to Article)
min. labelling: 15N, 13C
The magnetisation is passed from 1H to 15N and then to 13CO. From here it is transferred to 13Cα and the chemical shift is evolved. The magnetisation is then transferred back via 13CO to 15N and 1H for detection. The chemical shift is only evolved for the 1HN, the 15NH and the 13Cα, but not for the 13CO. This results in a spectrum which is like the HNCA, but which is selective for the Cα of the preceding residue.
This experiment can be useful for backbone assignment when used in conjunction with the HNCA, HNCO and HN(CA)CO if the CBCANNH and CBCA(CO)NNH spectra are of bad quality.
Top
CBCA(CO)NH / HN(CO)CACB (3D)
Reference:
S. Grzesiek and A. Bax (1992)
J. Am. Chem. Soc. 114 6291-6293. (
Link to Article)
min. labelling: 15N, 13C
Magnetisation is transferred from 1Hα and 1Hβ to 13Cα and 13Cβ, respectively, and then from 13Cβ to 13Cα. From here it is transferred first to13CO, then to 15NH and then to 1HN for detection. The chemical shift is evolved simultaneously on 13Cα and 13Cβ, so these appear in one dimension. The chemical shifts evolved in the other two dimensions are 15NH and 1HN. The chemical shift is not evolved on 13CO.
Along with the CBCANNH and HSQC this forms the standard set of experiments needed for backbone assignment. For large proteins the signal-to-noise may not be great and assignment using the HNCA, HN(CO)CA, HNCO and HN(CA)CO may form a better strategy. When using deuterated protein, the spectrum has to be recorded as an 'out-and-back' method and the signal-to-noise suffers even further.
Top
CBCANH / HNCACB (3D)
Reference:
S. Grzesiek and A. Bax (1992)
J. Magn. Reson. 99 201-207. (
Link to Article)
min. labelling: 15N, 13C
Magnetisation is transferred from 1Hα and 1Hβ to 13Cα and 13Cβ, respectively, and then from 13Cβ to 13Cα. From here it is transferred first to 15NH and then to 1HN for detection. Transfer form Cαi-1 can occur both to 15Ni-1 and 15Ni, or viewed the other way, magnetisation is transferred to 15Ni from both 13Cαi and 13Cαi-1. Thus for each NH group there are two Cα and Cβ peaks visible. The chemical shift is evolved simultaneously on 13Cα and 13Cβ, so these appear in one dimension. The chemical shifts evolved in the other two dimensions are 15NH and 1HN.
Along with the CBCA(CO)NNH and HSQC this forms the standard set of experiments needed for backbone assignment. For large proteins the signal-to-noise may not be great and assignment using the HNCA, HN(CO)CA, HNCO and HN(CA)CO may form a better strategy. When using deuterated protein, the spectrum has to be recorded as an 'out-and-back' method and the signal-to-noise suffers even further.
Top
CC(CO)NH (3D)
Reference:
S. Grzesiek, J. Anglister and A. Bax (1993)
J. Magn. Reson., Ser. B 101 114-119. (
Link to Article)
min. labelling: 15N, 13C
Magnetisation is transferred from the side-chain hydrogen nuclei to their attached 13C nuclei. Then isotropic 13C mixing is used to transfer magnetisation between the carbon nuclei. From here, magnetisation is transferred to the carbonyl carbon, on to the amide nitrogen and finally the amide hydrogen for detection. The chemical shift is evolved simultaneously on all side-chain carbon nuclei, as well as on the amide nitrogen and hydrogen nuclei, resulting in a three-dimensional spectrum.
This is a useful spectrum for obtaining carbon side-chain assignments, but isn't necessarily a must.
Top
H(CCO)NH (3D)
References:
G.T. Montelione, B.A. Lyons, S.D. Emerson and M. Tashiro (1992)
J. Am. Chem. Soc. 114 10974-10975. (
Link to Article)
S. Grzesiek, J. Anglister and A. Bax (1993)
J. Magn. Reson., Ser. B 101 114-119. (
Link to Article)
min. labelling: 15N, 13C
Magnetisation is transferred from the side-chain hydrogen nuclei to their attached 13C nuclei. Then isotropic 13C mixing is used to transfer magnetisation between the carbon nuclei. From here, magnetisation is transferred to the carbonyl carbon, on to the amide nitrogen and finally the amide hydrogen for detection. The chemical shift is evolved simultaneously on all side-chain hydrogen nuclei, as well as on the amide nitrogen and hydrogen nuclei, resulting in a three-dimensional spectrum with one nitrogen and two hydrogen dimensions.
This is a useful spectrum for obtaining hydrogen side-chain assignments, but isn't necessarily a must.
Top
HBHA(CO)NH (3D)
Reference:
S. Grzesiek and A. Bax (1993)
J. Biomol. NMR 3 185-204. (
Link to Article)
min. labelling: 15N, 13C
This experiment is similar to the CBCAC(CO)NH: magnetisation is transferred from 1Hα and 1Hβ to 13Cα and 13Cβ, respectively, and then from 13Cβ to 13Cα. From here it is transferred first to13CO, then to 15NH and then to 1HN for detection. The chemical shift it not evolved on any of the carbon atoms. Instead, it is evolved on the 1Hα and 1Hβ, the 15NH and 1HN. This results in a three-dimensional spectrum with one nitrogen and two hydrogen dimensions.
This is a useful spectrum for obtaining Hα and Hβ assignments, but isn't necessarily a must.
Top
HCCH-TOCSY (3D)
min. labelling: 15N, 13C
References:
A. Bax, G.M. Clore and A.M. Gronenborn (1990)
J. Magn. Reson. 88 425-431. (
Link to Article)
E.T. Olejniczak, R.X. Xu and S.W. Fesik (1992)
J. Biomol. NMR 2 655-659. (
Link to Article)
Magnetisation is transferred from the side-chain hydrogen nuclei to their attached 13C nuclei. This is followed by isotropic 13C mixing and finally transfer back to the side-chain hydrogen atoms for detection.
This spectrum is used for side-chain assignment. It yields strips at each carbon frequency in the side chain in which all side-chain hydrogen resonances are visible. The HCCH-COSY is a slightly less crowded version in which only the hydrogen resonances of the own and neighbouring carbons are visible. Note that the spectrum is usually displayed in such a way that the 1H detected dimension is shown along the y-axis rather than the x-axis. This means that the peaks belonging to one side-chain appear in a vertical strip rather than a horizontal strip.
Top
HCCH-COSY (3D)
References:
A. Bax, G.M. Clore, P.C. Driscoll, A.M.Gronenborn, M. Ikura and L.E. Kay (1990)
J. Magn. Reson. 87 620-627. (
Link to Article)
L.E. Kay, M. Ikura and A. Bax (1990)
J. Am. Chem. Soc. 112 888-889. (
Link to Article)
M. Ikura, L.E. Kay and A. Bax (1991)
J. Biomol. NMR 1 299-304. (
Link to Article)
min. labelling: 15N, 13C
Magnetisation is transferred from the side-chain hydrogen nuclei to their attached 13C nuclei. Magnetisation is then exchanged between neighbouring 13C nuclei via the J-coupling and finally transferred back to the side-chain hydrogen atoms for detection.
This spectrum can be useful in aiding side-chain assignment.
Top
15N-TOCSY-HSQC (3D)
References:
D. Marion, P.C. Driscoll, L.E. Kay, P.T. Wingfield, A. Bax, A.M. Gronenborn and G.M. Clore (1989)
Biochemistry 28 6150-6156. (
Link to Article)
min. labelleing: 15N
To start with, an isotropic mixing step transfers magnetisation between 1H spins. Then the magnetisation is transferred to neighbouring 15N nuclei and back to 1H for detection.
This can help identify amino acid types. If assignment is being done using 15N protein, only, it is a must. It can also help with the assignment of side-chain hydrogen atoms, though normally an HCCH-TOCSY or HCCH-COSY spectrum is necessary for unambiguous side-chain hydrogen assignment.
Top
13C-HMQC (2D)
References:
see
Protein NMR Spectroscopy - Principles and Practice by J. Cavanagh, W. Fairbrother, A.G. Palmer III and N.J. Skleton (Academic Press). (
Link to Publisher)
min. labelling: 13C
The magnetisation is transferred from 1H to 13C and then back again for detection and all 1H-13C moieties, regardless of chemical type, are observed.
This is the carbon equivalent of the 1H-15N-HSQC. All H-C correlations are shown. It can be useful as a basis for picking a 3D 13C NOESY spectrum. It can also be a useful reference for the aromatic peaks.
Top
H-H NOESY (2D)
References:
see
Protein NMR Spectroscopy - Principles and Practice by J. Cavanagh, W. Fairbrother, A.G. Palmer III and N.J. Skleton (Academic Press). (
Link to Publisher)
min. labelling: none
Magnetisation is exchanged between all hydrogens using the NOE.
This spectrum can be used both to help assignment (especially of aromatic residues) and to get structural restraints. It can get very crowded for large proteins, but can nonetheless provide some useful constraints for methyl and aromatic hydrogens. The quality can be improved by using D2O as the solvent, though this means that the exchangable NH protons will not be visible.
Top
15N-NOESY-HSQC (3D)
References:
D. Marion, P.C. Driscoll, L.E. Kay, P.T. Wingfield, A. Bax, A.M. Gronenborn and G.M. Clore (1989)
Biochemistry 28 6150-6156. (
Link to Article)
D. Marion, L.E. Kay, S.W. Sparks, D.A. Torchia and A. Bax (1989)
J. Am. Chem. Soc. 111 1515-1517. (
Link to Article)
E.R.P. Zuiderweg and S.W. Fesik (1989)
Biochemistry 28 2387-2391. (
Link to Article)
min. labelling: 15N
To start with, magnetisation is exchanged between all hydrogens using the NOE. Then the magnetisation is transferred to neighbouring 15N nuclei and back to 1H for detection.
This spectrum can be used to obtain restraints for structure calculations. In this case the NOESY mixing time should probably be around 80ms. It can also be used to help assignment, and for small to medium-sized proteins, assignment using this and 15N-TOCSY-HSQC only is possible. In this case it may be useful to use a slightly longer NOESY mixing time.
Top
13C-NOESY-HSQC (3D)
References:
D. Marion, P.C. Driscoll, L.E. Kay, P.T. Wingfield, A. Bax, A.M. Gronenborn and G.M. Clore (1989)
Biochemistry 28 6150-6156. (
Link to Article)
D. Marion, L.E. Kay, S.W. Sparks, D.A. Torchia and A. Bax (1989)
J. Am. Chem. Soc. 111 1515-1517. (
Link to Article)
E.R.P. Zuiderweg and S.W. Fesik (1989)
Biochemistry 28 2387-2391. (
Link to Article)
min. labelling: 13C
To start with, magnetisation is exchanged between all hydrogens using the NOE. Then the magnetisation is transferred to neighbouring 13C nuclei and back to 1H for detection. Transfer either occurs to/from the aliphatic 13C nuclei or to/from the aromatic 13C nuclei (but not both) depending on the 13C frequency used during the pulse sequence.
This spectrum is used to obtain restraints for structure calculations. It will usually be recorded with 100% D2O as the solvent - this minimises artefacts. This can be centred either on the aliphatic or on the aromatic carbons. The spectrum centred on the aromatic carbons can also be very useful for the assignment of the aromatic residues.
Top
13C-HMQC-NOESY (3D)
References:
S.W. Fesik and E.R.P. Zuiderweg (1988)
J. Magn. Reson. 78 588-593. (
Link to Article)
min. labelling: 13C
First there is an HMQC step in which magnetisation is transferred from 1H to 13C and back again. This is then followed by a NOESY step in which the magnetisation is transferred to any other hydrogen nucleus close by. This is followed by detection.
This spectrum is used to obtain restraints for structure calculations. It will usually be recorded with 100% D2O as the solvent - this minimises artefacts. Its advantage over the 13C-NOESY-HSQC, is that the NOESY dimension is the directly detected one, and therefore the resolution in the NOESY dimension is significantly increased. A further advantage, is that the aliphatic and aromatic carbon regions can both be included in one spectrum and the aromatic region can be extremely useful for the assignment of the aromatic residues.
