A pulsed field gradient isotope‐filtered 3D 13C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

A pulsed field gradient three‐dimensional isotope‐filtered 13C HMQC‐NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly 13C labeled trp‐repressor, its natural abundance co‐repressor, l‐tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra‐ and intermolecular NOEs in the isotope‐edited 3D 13C HMQC‐NOESY spectrum of the complex.

[1]  L. Kay,et al.  Three-dimensional NOESY-HMQC spectroscopy of a 13C-labeled protein , 1990 .

[2]  A. Bax,et al.  Optimized recording of heteronuclear multidimensional NMR spectra using pulsed field gradients , 1992 .

[3]  E. Olejniczak,et al.  An improved method for selectively observing protons attached to 12C in the presence of 1H13C spin pairs , 1992 .

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

[5]  Ad Bax,et al.  Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins , 1993, Journal of biomolecular NMR.

[6]  A. Gronenborn,et al.  Solution structure of a calmodulin-target peptide complex by multidimensional NMR. , 1994, Science.

[7]  Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor. , 1990, Biochemistry.

[8]  C. Yanofsky,et al.  High level production and rapid purification of the E. coli trp repressor. , 1986, Nucleic acids research.

[9]  Ad Bax,et al.  Methodological advances in protein NMR , 1993 .

[10]  L. Kay,et al.  An enhanced-sensitivity pure absorption gradient 4D 15N, 13C-edited NOESY experiment , 1993 .

[11]  A. Petros,et al.  Three-dimensional structure of the FK506 binding protein/ascomycin complex in solution by heteronuclear three- and four-dimensional NMR. , 1993, Biochemistry.

[12]  Ad Bax,et al.  Isotope-filtered 2D NMR of a protein-peptide complex: study of a skeletal muscle myosin light chain kinase fragment bound to calmodulin , 1992 .

[13]  G. Wider,et al.  Use of a double half-filter in two-dimensional proton NMR studies of receptor-bound cyclosporin , 1990 .

[14]  T. Pawson,et al.  Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-γ1 complexed with a high affinity binding peptide , 1994, Cell.

[15]  S. Grzesiek,et al.  Isotope-Filtered 2D HOHAHA Spectroscopy of a Peptide-Protein Complex Using Heteronuclear Hartmann-Hahn Dephasing , 1994 .

[16]  A M Gronenborn,et al.  NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1. , 1993, Science.

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

[18]  T. Logan,et al.  A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments , 1993, Journal of biomolecular NMR.

[19]  M Nilges,et al.  The solution structure of the Tyr41-->His mutant of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13. , 1994, Journal of molecular biology.

[20]  G. Wagner,et al.  Selective excitation of 1H resonances coupled to 13C. Hetero COSY and RELAY experiments with 1H detection for a protein , 1986 .

[21]  C. Hilbers,et al.  Overcoming the ambiguity problem encountered in the analysis of nuclear overhauser magnetic resonance spectra of symmetric dimer proteins , 1993 .

[22]  A M Gronenborn,et al.  Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy. , 1991, Science.