A 1H-NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective 2H-labeling. Complete assignment and structural analysis of the aromatic resonances for a 22-kDa homodimer.
1H-NMR experiments have been performed on transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPO1. To study this 22-kDa homodimeric DNA-binding protein, a selective 2H-labeling 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 2H-labeled variants, as opposed to the fully 1H-containing 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 beta-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, 84-90)] 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 T1 relaxation times of the aromatic resonances for the fully 1H-containing protein and the relaxation time enhancements upon selective 2H-labeling, respectively.
Reisman
J M
JM
Department of Chemistry, University of California, San Diego.
Hsu
V L
VL
Jariel-Encontre
I
I
Lecou
C
C
Sayre
M H
MH
Kearns
D R
DR
Parello
J
J
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry; D001435:Bacteriophages / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry; D003903:Deuterium; D006859:Hydrogen; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D016329:Sp1 Transcription Factor / Q000737:chemistry*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016329:Sp1 Transcription Factor; C040330:histone-like protein HU, bacteria; D006859:Hydrogen; D003903:Deuterium