Abstract
Full automation of the analysis of spectra is a prerequisite for high-throughput NMR studies in structural or functional genomics. Sequence-specific assignments often form the major bottleneck. Here, we present a procedure that yields nearly complete backbone and side chain resonance assignments starting from a set of heteronuclear three-dimensional spectra. Neither manual intervention, e.g., to correct lists obtained from peak picking before feeding these to an assignment program, nor protein-specific information, e.g., structures of homologous proteins, were required. By combining two earlier published procedures, AUTOPSY [Koradi et al. (1998) J. Magn. Reson., 135, 288–297] and GARANT [Bartels et al. (1996) J. Biomol. NMR, 7, 207–213], with a new program, PICS, all necessary steps from spectra analyses to sequence-specific assignments were performed fully automatically. Characteristic features of the present approach are a flexible design allowing as input almost any combination of NMR spectra, applicability to side chains, robustness with respect to parameter choices (such as noise levels) and reproducibility. In this study, automated resonance assignments were obtained for the 14 kD blue copper protein azurin from P. aeruginosa using five spectra: HNCACB, HNHA, HCCH-TOCSY, 15N-NOESY-HSQC and 13C-NOESY-HSQC. Peaks from these three-dimensional spectra were filtered and calibrated with the help of two two-dimensional spectra: 15N-HSQC and 13C-HSQC. The rate of incorrect assignments is less than 1.5% for backbone nuclei and about 3.5% when side chain protons are also considered.
Similar content being viewed by others
References
Atreja, H.S., Sahu, S.C., Chary, K.V.R. and Govil, G. (2000) J. Biomol. NMR, 17, 125-136.
Bartels, C., Billeter, M., Güntert, P. and Wüthrich, K. (1996) J. Biomol. NMR, 7, 207-213.
Bartels, C., Güntert, P., Billeter, M. and Wüthrich, K. (1997) J. Comput. Chem., 18, 139-149.
Bartels, C., Xia, T.H., Billeter, M., Güntert, P. and Wüthrich, K. (1995) J. Biomol. NMR, 6, 1-10.
Billeter, M. (1991) In Computational Aspects of the Study of Biological Macromolecules by Nuclear Magnetic Resonance Spectroscopy, Vol. 255: NATO ASI Series, Hoch J.C., Poulsen F.M. and Redfield C. (Eds.) Plenum, New York, pp. 279-290.
Billeter, M., Basus, V.J. and Kuntz, I.D. (1988) J. Magn. Reson., 76, 400-415.
Buchler, N.E.G., Zuiderweg, E.R.P., Wang, H. and Goldstein, R.A. (1997) J. Magn. Reson., 125, 34-42.
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277-293.
Gronwald, W., Moussa, S., Elsner, R., Jung, A., Ganslmeier, B., Trenner, J., Kremer, W., Neidig, K.P. and Kalbitzer, H.R. (2002) J. Biomol. NMR, 23, 271-287.
Gronwald, W., Willard, L., Jellard, T., Boyko, R.F., Rajarathnam, K., Wishart, D.S., Sönnichsen, F.D. and Sykes, B.D. (1998) J. Biomol. NMR, 12, 395-405.
Güntert, P. (1998) Quart. Rev. Biophys., 31, 145-237.
Güntert, P., Salzmann, M., Braun, D. and Wüthrich, K. (2000) J. Biomol. NMR, 18, 129-137.
Hajduk, P.J., Meadows, R.P. and Fesik, S.W. (1999) Quart. Rev. Biophys., 32, 211-240.
Herrmann, T., Güntert, P. and Wüthrich, K. (2002) J. Mol. Biol., 319, 209-227.
Härd, T. (1999) Quart. Rev. Biophys., 32, 57-98.
Koradi, R., Billeter, M., Engeli, M., Güntert, P. and Wüthrich, K. (1998) J. Magn. Reson., 135, 288-297.
Korzhnev, D.M., Billeter, M., Arseniev, A.S. and Orekhov, V.Y. (2001) Prog. Nucl. Magn. Reson. Spectrosc., 38, 197-266.
Leckner, J. (2001) Folding and Structure of Azurin - The Influence of a Metal, Chalmers University of Technology, Göteborg, Sweden.
Leutner, M., Gschwind, R.M., Liermann, J., Schwarz, C., Gemmecker, G. and Kessler, H. (1998) J. Biomol. NMR, 11, 31-43.
Li, K.B. and Sanctuary, B.C. (1997a) J. Chem. Inf. Comput. Sci., 37, 359-366.
Li, K.B. and Sanctuary, B.C. (1997b) J. Chem. Inf. Comput. Sci., 37, 467-477.
Linge, J.P., O'Donoghue, S.I. and Nilges, M. (2001) Meth. Enzymol., 339, 71-90.
Lukin, J.A., Gove, A.P., Talukdar, S.N. and Ho, C. (1997) J. Biomol. NMR, 9, 151-166.
Moseley, H.N. and Montelione, G.T. (1999) Curr. Opin. Struct. Biol., 9, 635-642.
Moseley, H.N., Monleon, D. and Montelione, G.T. (2001) Meth. Enzymol., 339, 91-108.
Tian, F., Valafar, H. and Prestegard, J.H. (2001) J. Amer. Chem. Soc., 123, 11791-11796.
Vuister, G.W. and Bax, A (1993) J. Amer. Chem. Soc., 115, 7772-7777.
Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley, New York, NY.
Zimmerman, D.E., Kulikowski, C.A., Huang, Y., Feng, W., Tashiro, M., Shimotakahara, S., Chien, C., Powers, R. and Montelione, G.T. (1997) J. Mol. Biol., 269, 592-610.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Malmodin, D., Papavoine, C.H. & Billeter, M. Fully automated sequence-specific resonance assignments of hetero- nuclear protein spectra. J Biomol NMR 27, 69–79 (2003). https://doi.org/10.1023/A:1024765212223
Issue Date:
DOI: https://doi.org/10.1023/A:1024765212223