Summary
Improved experimental schemes for the recently introduced J-modulated [15N,1H]-correlation experiment for measurements of the homonuclear amide proton-Cα proton vicinal coupling constants.3JHNα, in uniformly15N-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of3JHNα. The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M=7.3 kDa), where at 13 C precise values of3JHNα in the range 2.0–9.5 Hz were obtained for all residues with resolved15N-1H cross peaks. It was then applied to theAntennapedia homeodomain complexed to a synthetic 14-base pair DNA fragment (molecular weight of the complex ∼ 18 kDa). The3JHNα values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.
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Abbreviations
- NOE:
-
nuclear Overhauser effect
- COSY:
-
two-dimensional correlated spectroscopy
- 3JHNα or J:
-
homonuclear vicinal amide proton-Cα proton coupling constant
- 434 repressor(1–69):
-
N-terminal DNA-binding domain of the 434 repressor comprising 69 residues
References
Anderson, J., Ptashne, M. and Harrison, S.C. (1984).Proc. Natl. Acad. Sci. USA.181, 1307–1311.
Arseniev, A., Schultze, P., Wörgötter, E., Braun, W., Wagner, G., Vasak, M., Kägi, J.H.R. and Wüthrich, K. (1988)J. Mol. Biol. 201, 637–657.
Bodenhausen, G. and Ruben, D. (1980)Chem. Phys. Lett.,69, 185–188.
Bystrov, V.F. (1976)Prog. NMR Spectrosc.,10, 41–81.
De Marco, A. and Wüthrich, K. (1976).J. Magn. Reson.,24, 201–204.
De Marco, A., Llinás, M., and Wüthrich, K. (1978).Biopolymers.17, 637–650.
Eccles, C., Güntert, P., Billeter, M., and Wüthrich, K. (1991)J. Biomol. NMR,1, 111–130.
Goldman, M. (1984).J. Magn. Reson. 60, 437–452.
Guéron, M., Leroy, J.L. and Griffey, R.H. (1983).J. Am. Chem. Soc. 105, 7262–7266.
Güntert, P., Braun, W., Billeter, M., and Wüthrich, K. (1989).J. Am. Chem. Soc.,111, 3997–4004.
Karplus, M. (1959).J. Phys. Chem. 30, 11–15.
Karplus, M. (1963)J. Am. Chem. Soc.,85, 2870–2871.
Kay, L.E. and Bax, A. (1990)J. Magn. Reson.,86, 110–126.
Kay, L.E., Brooks, B., Sparks, S.W., Torchia, D.A. and Bax, A. (1989)J. Am. Chem. Soc. 111, 5488–5490.
Kim, Y., and Prestegard, J.H. (1989)J. Magn. Reson. 84, 9–13.
Ludvigsen, S., Andersen, K.V. and Poulsen, F.M. (1991)J. Mol. Biol. 217, 731–736.
McIntosh, L. and Dahlquist, F.W. (1990)Quart. Rev. Biophys.,23, 1–38.
Marion, D. and Wüthrich, K. (1983)Biochem. Biophys. Res. Commun. 113, 967–974.
Messerle, B.A., Wider, G., Otting, G., Weber, C. and Wüthrich, K. (1989)J. Magn. Reson.,85, 608–613.
Montelione, G. and Wagner, G. (1989)J. Am. Chem. Soc. 111, 5474–5475.
Nagayama, K. and Wüthrich, K. (1981)Eur. J. Biochem.,115, 653–657.
Neri, D., Szyperski, T., Otting, G., Senn, H. and Wüthrich, K. (1989)Biochemistry,28, 7510–7516.
Neri, D., Otting, G. and Wüthrich, K. (1990).J. Am. Chem. Soc. 112, 3663–3665.
Neri, D., Billeter, M. and Wüthrich, K. (1992)J. Mol. Biol.,223, 743–767.
Neuhaus, D., Wagner, G., Vasak, M., Kägi, J.H.R. and Wüthrich, K. (1985)Eur. J. Biochem.,151, 257–273.
Otting, G. and Wüthrich, K. (1988)J. Magn. Reson.,76, 569–574.
Otting, G. and Wüthrich, K. (1990)Quart. Rev. Biophys. 23, 39–96.
Otting, G., Qian, Y.Q., Billeter, M., Müller, M., Affolter, M., Gehring, W.J. and Wüthrich, K. (1990)EMBO J.,9, 3085–3092.
Pardi, A., Billeter, M. and Wüthrich, K. (1984).J. Mol. Biol. 180, 741–751.
Press, W.M., Flannery, B.P., Teukolsky, S.A. and Vetterling, W.T. (1986).Numerical Recipes, Cambridge University Press, Cambridge.
Qian, Y.Q., Billeter, M., Otting, G., Müller, M., Gehring, W.J. and Wüthrich, K. (1989)Cell,59, 573–580.
Shaka, A.J., Barker, P.B., Bauer, C.J. and Freeman, R. (1986).J. Magn. Reson. 67, 396–401.
Sørensen, O.W., Eich, G.W., Levitt, M.H., Bodenhausen, G. and Ernst, R.R. (1983)Prog. NMR Spectrosc.,16, 163–192.
Wagner, G., Schmieder, P. and Thanabal, V. (1991)J. Magn. Reson. 93, 436–440.
Weber, C., Wider, G., von Freyberg, B., Traber, R., Braun, W., Widmer, H. and Wüthrich, K. (1991)Biochemistry,30, 6563–6574.
Wider, G., Neri, D., Otting, G. and Wüthrich, K. (1989)J. Magn. Reson.,83, 426–431.
Wüthrich, K. (1986)NMR of Proteins and Nucleic Acids, Wiley, New York.
Wüthrich, K. (1990)J. Biol. Chem.,265, 22059–22062.
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Billeter, M., Neri, D., Otting, G. et al. Precise vicinal coupling constants3JHNα in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments. J Biomol NMR 2, 257–274 (1992). https://doi.org/10.1007/BF01875320
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DOI: https://doi.org/10.1007/BF01875320