N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38(2001)83-114 repressor bound to a 20 base pair palindromic DNA operator)was determined by recording homonuclear G4T11 2D and 3D spectra for complexes with different T120 0 1 deuterium labeled trp repressor analogs as well as 140 heteronuclear spectra for complexes with uniformly SN, C-labeled trp repressor[29] 145 The use of perdeuterated protein in H2O (i.e. >90% "H incorporation at nonlabile positions and about 90% of labile positions protonated) led to the assignments A1400A7 of almost all backbone and C resonances of the 155 37 kDa trp repressor-operator DNA complex [30 and of a 64 kDa repressor-operator complex(two Indem dimers bound to a 22 base pair symmetric tryptophan)[31, 32 Samples of perdeuterated protein containing selec A7/A80 tive protonated or N,C, H labeled residues are HB/H6 A110 used to characterize specific contacts between the protein and the DNA. For example in the study of 13C the dna binding domain of the transcription factor NFATCI bound to a 12 base pair DNA, Zhou and coworkers [33] performed 2D H-H homonuclear 150 A11 NOESY experiment on complexes containing perdeuterated protein with fully protonated Tyr and 1551 Phe residues to characterize the contacts between Tyr ppm 442 and dnA. These authors also mentioned the use of site-specific deuteration at C2 of Ade6 to confirm the close proximity of Arg555 and Ade6 Fig 4 Portion of H-C HSQC spectra at 298K in D O, showi the correlations between aromatic protons and carbons of a 15 base pair DNA containing the binding site of NHP6. Upper spectrum 2.6. Transverse relaxation-optimized spectroscopy ple of C, N 15-mer DNA with upper strand labeled only (TROSY Lower spectrum: sample of C, N 15-mer DNA with lower strand abeled only(adapted from Fig. 8 of Ref. [271). Reprinted with the Recently, wuthrich and coworkers have proposed a permission of J Feigon and of Oxford University Press(O 1999) lew approach to reduce significantly transverse relaxation rates in multidimensional NMR experi- 2.5. Deuteration ments and thus eliminate one of the obstacles to the study of large molecules and complexes by NMR In the case of large protein-DNA complexes, the [34-36] conventional backbone triple resonance experiments The relaxation of backbone N nuclei is are unsuccessful for providing complete assignment dominated by the interaction between N of the protein resonances. Therefore, selective proto- nuclei and its directly attached proton and by the on and/or uniform complete or fractional de chemical shift sotropy interaction. As the N tion in combination or not withC, N-labeling of the CSA tensor is nearly axially symmetric and has its protein are used to simplify proton spectra( Fig 4)and axis making a small angle with the N-H bond vector, to overcome the problem of rapid transverse nuclear theN nuclei will have a relaxation rate depending on pin relaxation[28] the spin state of the proton attached to it. TROSY uses The structure of a 37 kDa trp repi this differential relaxation to select only the compo- DNA complex(homodimeric 107 residue E. coli trp nent which relaxes the more slowly. Using this2.5. Deuteration In the case of large protein±DNA complexes, the conventional backbone triple resonance experiments are unsuccessful for providing complete assignment of the protein resonances. Therefore, selective proto￾nation and/or uniform complete or fractional deutera￾tion in combination or not with 13C,15N-labeling of the protein are used to simplify proton spectra (Fig. 4) and to overcome the problem of rapid transverse nuclear spin relaxation [28]. The structure of a 37 kDa trp repressor±operator DNA complex (homodimeric 107 residue E. coli trp repressor bound to a 20 base pair palindromic DNA operator) was determined by recording homonuclear 2D and 3D spectra for complexes with different deuterium labeled trp repressor analogs as well as heteronuclear spectra for complexes with uniformly 15N,13C-labeled trp repressor [29]. The use of perdeuterated protein in H2O (i.e. .90% 2 H incorporation at nonlabile positions and about 90% of labile positions protonated) led to the assignments of almost all backbone and Cb resonances of the 37 kDa trp repressor±operator DNA complex [30] and of a 64 kDa repressor±operator complex (two tandem dimers bound to a 22 base pair symmetric DNA operator and the corepressor analog 5-methyl￾tryptophan) [31,32]. Samples of perdeuterated protein containing selec￾tive protonated or 15N,13C,1 H labeled residues are used to characterize speci®c contacts between the protein and the DNA. For example in the study of the DNA binding domain of the transcription factor NFATC1 bound to a 12 base pair DNA, Zhou and coworkers [33] performed 2D 1 H±1 H homonuclear NOESY experiment on complexes containing perdeuterated protein with fully protonated Tyr and Phe residues to characterize the contacts between Tyr 442 and DNA. These authors also mentioned the use of site-speci®c deuteration at C2 of Ade6 to con®rm the close proximity of Arg555 and Ade6. 2.6. Transverse relaxation-optimized spectroscopy (TROSY) Recently, WuÈthrich and coworkers have proposed a new approach to reduce signi®cantly transverse relaxation rates in multidimensional NMR experi￾ments and thus eliminate one of the obstacles to the study of large molecules and complexes by NMR [34±36]. The relaxation of peptide backbone 15N nuclei is dominated by the dipolar interaction between 15N nuclei and its directly attached proton and by the chemical shift anisotropy interaction. As the 15N CSA tensor is nearly axially symmetric and has its axis making a small angle with the N±H bond vector, the 15N nuclei will have a relaxation rate depending on the spin state of the proton attached to it. TROSY uses this differential relaxation to select only the compo￾nent which relaxes the more slowly. Using this N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 89 Fig. 4. Portion of 1 H±13C HSQC spectra at 298 K in D2O, showing the correlations between aromatic protons and carbons of a 15 base pair DNA containing the binding site of NHP6. Upper spectrum: sample of 13C,15N 15-mer DNA with upper strand labeled only. Lower spectrum: sample of 13C,15N 15-mer DNA with lower strand labeled only (adapted from Fig. 8 of Ref. [27]). Reprinted with the permission of J. Feigon and of Oxford University Press (q 1999)