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Precise vicinal coupling constants3JHNα in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments

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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

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Authors and Affiliations

  1. Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule-Hönggerberg, CH-8093, Zürich, Switzerland

    Martin Billeter, Dario Neri, Gottfried Otting, Yan Qiu Qian & Kurt Wüthrich

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  1. Martin Billeter
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  2. Dario Neri
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  3. Gottfried Otting
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  4. Yan Qiu Qian
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  5. Kurt Wüthrich
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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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