Revision history - NMR Wiki Q&A Forum

Revision history [back]

initial version

posted May 25 '13 at 14:13

To compliment the earlier answers -- The overall sensitivity of a 3D experiment depends on the product of the efficiency of coherence transfer of each of the steps. For HNCO, we need to consider the efficiency of coherence transfer pathways from 1H to 15N and from 15N to 13C of carbonyl (and back). The efficiency of a coherence transfer process depends on the ratio of the magnitude of the J-coupling to the line-width; in addition other available nonproductive/undesirable (branching) coherence transfer paths would lower the sensitivity of the experiment. Hence, in addition to the magnitude of the coupling constant, (discussed in earlier posts), we also need to consider the linewidths. The linewidths of the carbonyl carbons are substantially lower than that of the linewidths of Ca atoms. This is yet another reason for the much higher sensitivity of HNCO compared to HNCA. The larger linewidth of the Ca is due to relaxation induced by attached hydrogen (Ha). The carbonyl carbon in the peptides/proteins is not covalently bonded to any hydrogen and hence a major relaxation mechanism is absent, resulting in a narrow linewidth. A long range HNCO was used to observe 0.2 Hz 3J-NC' coupling in hydrogen bonded GG base pairs - this was possible due to the favorable relaxation properties of the system. Yet another reason for the higher sensitivity of HNCO compared to HNCA is due the presence of multiple coherence transfer pathways originating from 15N in the HNCA for proteins. Coherence transfer occurs from 15N to Ca of the same residue as well as to the Ca of the previous residue, due to the relatively small difference in the coupling constants from 15N to the Ca of same and previous residues - this results in a reduction in sensitivity. Furthermore, the unresolved coupling of Ca to Cb of side-chain may become effective if the resolution (and hence acquisition time) in the carbon dimension increases. Since the carbonyl carbon does not have significant J-coupling to any other carbon atom with similar chemical shift, such alternative coherence transfer pathways are minimized in the HNCO experiments on proteins.

No.1 Revision

posted May 25 '13 at 14:20

sekhar Talluri
621

To compliment the earlier answers --

The overall sensitivity of a 3D experiment depends on the product of the efficiency of coherence transfer of each of the steps. For HNCO, we need to consider the efficiency of coherence transfer pathways from 1H to 15N and from 15N to 13C of carbonyl (and back). The efficiency of a coherence transfer process depends on the ratio of the magnitude of the J-coupling to the line-width; in addition other available nonproductive/undesirable (branching) coherence transfer paths would lower the sensitivity of the experiment.

Hence, in addition to the magnitude of the coupling constant, (discussed in earlier posts), we also need to consider the linewidths. The linewidths of the carbonyl carbons are substantially lower than that of the linewidths of Ca atoms. This is yet another reason for the much higher sensitivity of HNCO compared to HNCA. The larger linewidth of the Ca is due to relaxation induced by attached hydrogen (Ha). The carbonyl carbon in the peptides/proteins is not covalently bonded to any hydrogen and hence a major relaxation mechanism is absent, resulting in a narrow linewidth.
A long range HNCO was used to observe 0.2 Hz 3J-NC' coupling in hydrogen bonded GG base pairs - this was possible due to the favorable relaxation properties of the ~~system.~~system. (Grzesiek, JBNMR, 2000, 16, 279; Chap 9, BioNMR in Drug research, Wiley-VCH)

Yet another reason for the higher sensitivity of HNCO compared to HNCA is due the presence of multiple coherence transfer pathways originating from 15N in the HNCA for proteins. Coherence transfer occurs from 15N to Ca of the same residue as well as to the Ca of the previous residue, due to the relatively small difference in the coupling constants from 15N to the Ca of same and previous residues - this results in a reduction in sensitivity. Furthermore, the unresolved coupling of Ca to Cb of side-chain may become effective if the resolution (and hence acquisition time) in the carbon dimension increases. Since the carbonyl carbon does not have significant J-coupling to any other carbon atom with similar chemical shift, such alternative coherence transfer pathways are minimized in the HNCO experiments on proteins.