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Sequence-specific 1H and 15N NMR1 assignments are reported for the transcription factor 1 (TF1), a 22-kDa type II DNA-binding protein (DBPII) that consists of two 99-residue monomers. An assignment strategy is employed that uses six complementary selectively deuterium-labeled TF1 variants and an uniformly 15N-labeled TF1 variant. Two-dimensional and three-dimensional homonuclear and heteronuclear NMR correlated spectra are analyzed and yield nearly complete assignments for the 1H and 15N resonances. Discrete protein secondary structure domains are also defined; in each monomer, three alpha-helices, an antiparallel beta-sheet, and an antiparallel beta-ribbon are identified. Analyses of two dimers formed from two distinct selectively deuteriated monomers serve to identify a number of interproton contacts as either intermonomeric or intramonomeric. An analysis of amide proton exchange reveals that the carboxy-terminal alpha-helix is less stable than the other two alpha-helices in each monomer. A previously proposed working structural model of the TF1 dimer [Geiduschek et al. (1990) J. Struct. Biol. 104, 84-90], based on the crystal structure of a highly homologous DBPII, the Bacillus stearothermophilus-encoded HU protein, is generally supported by our results. Several departures from this model, however, are noted. Most notably, the carboxy-terminal tail of TF1 adopts an alpha-helical conformation with a backbone distortion at Lys93.

'H-NMR experiments have been performed on transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPOl. To study this 22-kDa homodimeric DNA-binding protein, a selective 'Hlabeling strategy has been employed. Complete sequence-specific assignments of all the resonances from the five aromatic residues were determined by a modified standard sequential-assignment procedure. The reduced contribution of spin diffusion upon the long-mixing-time nuclear-overhauser-enhancement spectroscopy for the selectively 'H-labeled variants, as opposed to the fully 'Hcontaining protein, has allowed for the identification of the spin systems and of the long-range dipolar contacts between Phe28 and Phe47 protons in the protein core and between Phe61 and Phe97 protons. The latter suggests an interaction between the proposed P-ribbon DNA-binding arm and the carboxy terminus of the paired monomer. A previously proposed TF1 structural model [Geiduschek, E. P., Schneider, G. J. & Sayre, M. H. (1990) J. Struct. Biol. 104,[84][85][86][87][88][89][90]] has been modified using constrained-energy-minimization calculations incorporating the experimentally determined set of aromatic-to-aromatic contacts. This new model has been analyzed with regard to the relative mobility and the relative solvent accessibility of the aromatic residues which have been measured by the nonselective T, relaxation times of the aromatic resonances for the fully 'Hcontaining protein and the relaxation time enhancements upon selective 2H-labeling, respectively.

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