Summary
A new triple-resonance 3D HNCOCA pulse scheme is presented, designed to identify the backbone nuclei (HN, N, CO, Cα) of doubly labelled proteins. The two carbon frequencies are labelled along the same indirect dimension and the corresponding dwell times can be independently scaled in order to account for the relaxation properties and chemical shift ranges of the CO and Cα. If one takes advantage of the symmetry properties of the spectra in the course of the peak picking, this 3D scheme has the same sensitivity as the 4D experiment, but with an improved resolution. The sequence is illustrated on a 0.5 mM sample of Rhodobacter capsulatus cytochrome c′ a homodimeric paramagnetic protein of 2×14 kDa. A resonance assignment strategy, based on a low-concentration 13C/15N-labelled sample and a more concentrated 15N-labelled sample, is proposed for proteins where the expression system shows a limited efficiency.
This is a preview of subscription content, log in via an institution to check access.
References
Bax, A., Mehlkopf, A.F. and Smidt, J. (1979) J. Magn. Reson, 35, 167–169.
Brutscher, B., Simorre, J.-P., Caffrey, M.S. and Marion, D. (1994) J. Magn. Reson. Ser. B, 105, 77–82.
Caffrey, M.S., Brutscher, B., Simorre, J.-P., Fitch, J., Cusanovich, M.A. and Marion, D. (1994) Eur. J. Biochem. 221, 63–75.
Caffrey, M.S., Simorre, J.-P., Brutscher, B., Cusanovich, M.A. and Marion, D. (1995) Biochemistry, in press.
Grzesiek, S. and Bax, A. (1992) J. Magn. Reson., 96, 432–440.
Ikura, M., Kay, L.E. and Bax, A. (1990) Biochemistry, 29, 4659–4667.
Kay, L.E., Ikura, M., Tschudin, R. and Bax, A. (1990) J. Magn. Reson., 89, 496–514.
Madsen, J.C. and Sørensen, O.W. (1992) J. Magn. Reson., 100, 431–436.
Marion, D., Ikura, M., Tschudin, R. and Bax, A. (1989) J. Magn. Reson., 85, 393–399.
Montelione, G.T. and Wagner, G. (1990) J. Magn. Reson., 87, 183–188.
Morelle, N., Brutscher, B., Simorre, J.-P. and Marion, D. (1995) J. Biomol. NMR, 5, 154–160.
Richarz, R. and Wüthrich, K. (1978) Biopolymers, 17, 2133–2141.
Simorre, J.-P., Brutscher, B., Caffrey, M.S. and Marion, D. (1994) J. Biomol. NMR, 4, 325–333.
Szypersky, T., Wider, G., Bushweller, J.H. and Wüthrich, K. (1993a) J. Am. Chem. Soc., 115, 9307–9308.
Szypersky, T., Wider, G., Bushweller, J.H. and Wüthrich, K. (1993b) J. Biomol. NMR, 3, 127–132.
VanDoren, S. and Zuiderweg, E.R.P. (1994) J. Magn. Reson. Ser. B, 104, 171–180.
Wishart, D. and Sykes, B.D. (1994) J. Biomol. NMR, 4, 127–333.
Zhu, G. and Bax, A. (1990) J. Magn. Reson., 90, 405–410.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Brutscher, B., Cordier, F., Simorre, JP. et al. High-resolution 3D HNCOCA experiment applied to a 28 kDa paramagnetic protein. J Biomol NMR 5, 202–206 (1995). https://doi.org/10.1007/BF00208811
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00208811