Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C′) and 3J(Hβ,C′) coupling constants in Desulfovibrio vulgaris flavodoxin

SummaryA simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings 3J(HN,C′) and 3J(Hβ,C′) in uniformly 13C-enriched proteins. The protocols for measuring accurate coupling constants are based on 1H,13C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95–105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of ϕ and X1 dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant 13C, 15N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 3J(HN,C′) and 100 3J(Hβ,C′) coupling constants are obtained from a single spectrum.

[1]  H. Kessler,et al.  SKALARE KOPPLUNGEN : IHRE ANALYSE UND IHRE VERWENDUNG ZUR STRUKTURAUFKLARUNG , 1995 .

[2]  L. Mueller,et al.  Nonresonant effects of frequency-selective pulses , 1992 .

[3]  P E Wright,et al.  Use of chemical shifts and coupling constants in nuclear magnetic resonance structural studies on peptides and proteins. , 1994, Methods in enzymology.

[4]  A. J. Shaka,et al.  Computer-optimized decoupling scheme for wideband applications and low-level operation , 1985 .

[5]  S. Grzesiek,et al.  Measurement of homo- and heteronuclear J couplings from quantitative J correlation. , 1994, Methods in enzymology.

[6]  J. Fox,et al.  The amino acid sequence of Desulfovibrio vulgaris flavodoxin. , 1977, Biochemical and biophysical research communications.

[7]  H. Rüterjans,et al.  Nuclear magnetic resonance studies of the old yellow enzyme. 1. 15N NMR of the enzyme recombined with 15N-labeled flavin mononucleotides. , 1985, European journal of biochemistry.

[8]  S. Glaser,et al.  Frequency-selective decoupling with recursively expanded soft pulses in multinuclear NMR , 1992 .

[9]  G. Bodenhausen,et al.  Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy , 1980 .

[10]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[11]  L. Mueller,et al.  Selective shaped pulse decoupling in NMR: homonuclear [carbon-13]carbonyl decoupling , 1992 .

[12]  R. R. Ernst,et al.  Correlation of connected transitions by two‐dimensional NMR spectroscopy , 1986 .

[13]  Torsion angles in the cystine bridge of oxytocin in aqueous solution. Measurements of circumjacent vicinal couplings between proton, carbon-13, and nitrogen-15 , 1980 .

[14]  Ad Bax,et al.  Investigation of complex networks of spin-spin coupling by two-dimensional NMR , 1981 .

[15]  M. Karplus.  Contact Electron‐Spin Coupling of Nuclear Magnetic Moments , 1959 .

[16]  R. R. Ernst,et al.  Practical aspects of the E.COSY technique. Measurement of scalar spin-spin coupling constants in peptides , 1987 .

[17]  Kurt Wüthrich,et al.  Application of ω1-decoupled 2D correlation spectra to the study of proteins , 1984 .

[18]  R. Fletcher,et al.  A New Approach to Variable Metric Algorithms , 1970, Comput. J..

[19]  Horst Kessler,et al.  An improved technique for correlating backbone amide protons with 15N and Hα protons (HN(CA)H) in isotopically enriched proteins , 1992 .

[20]  M. Johnson.  Evaluation and propagation of confidence intervals in nonlinear, asymmetrical variance spaces. Analysis of ligand-binding data. , 1983, Biophysical journal.

[21]  R. Sokal,et al.  Biometry.@@@Statistical Tables. , 1982 .

[22]  G. Harbison.  Interference between J-couplings and cross-relaxation in solution NMR spectroscopy : consequences for macromolecular structure determination , 1993 .

[23]  H. Schwalbe,et al.  Conformation of valine side chains in ribonuclease T1 determined by NMR studies of homonuclear and heteronuclear 3J coupling constants. , 1994, Biochemistry.

[24]  K. Wüthrich,et al.  Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. , 1983, Biochemical and biophysical research communications.

[25]  Geoffrey Bodenhausen,et al.  Gaussian pulse cascades: New analytical functions for rectangular selective inversion and in-phase excitation in NMR , 1990 .

[26]  D. Shanno.  Conditioning of Quasi-Newton Methods for Function Minimization , 1970 .

[27]  E. S. Pearson,et al.  TABLES OF PERCENTAGE POINTS OF THE INVERTED BETA (F) DISTRIBUTION , 1943 .

[28]  Martin Karplus,et al.  Vicinal Proton Coupling in Nuclear Magnetic Resonance , 1963 .

[29]  K D Watenpaugh,et al.  Comparison of the crystal structures of a flavodoxin in its three oxidation states at cryogenic temperatures. , 1993, Journal of molecular biology.

[30]  A. Bax,et al.  Reparametrization of the Karplus Relation for 3J(H.alpha.-N) and 3J(HN-C') in Peptides from Uniformly 13C/15N-Enriched Human Ubiquitin , 1995 .

[31]  G. Montelione,et al.  A general approach for determining scalar coupling constants in polypeptides and proteins , 1992, Biopolymers.

[32]  Ad Bax,et al.  Baseline distortion in real-Fourier-transform NMR spectra , 1988 .

[33]  S. Macura,et al.  Hydrogen Bonding Networks in Proteins As Revealed by the Amide 1JNC' Coupling Constant , 1995 .

[34]  G. Montelione,et al.  2D and 3D HCCH TOCSY experiments for determining 3J(HαHβ) coupling constants of amino acid residues , 1992 .

[35]  G. Curley,et al.  Homonuclear and heteronuclear NMR studies of oxidized Desulfovibrio vulgaris flavodoxin. Sequential assignments and identification of secondary structure elements. , 1993, European journal of biochemistry.

[36]  G. Wagner,et al.  1H and 15N resonance assignments of oxidized flavodoxin from Anacystis nidulans with 3D NMR. , 1991, Biochemistry.

[37]  A. Bax,et al.  An empirical correlation between 1JC.alpha.H.alpha. and protein backbone conformation , 1992 .

[38]  D. Goldfarb.  A family of variable-metric methods derived by variational means , 1970 .

[39]  S. Ghisla,et al.  Mechanisms of flavoprotein-catalyzed reactions. , 1989, European journal of biochemistry.

[40]  William H. Press,et al.  Numerical recipes , 1990 .

[41]  L. Mueller,et al.  Coherence quenching induced by frequency-selective homonuclear decoupling , 1992 .

[42]  Ad Bax,et al.  Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins , 1989 .