CONFORMATIONS AND CONFORMATIONAL-CHANGES OF 4 PHE-]TRP VARIANTS OF THE DNA-BINDING HISTONE-LIKE PROTEIN, HBSU, FROM BACILLUS-SUBTILIS STUDIED BY CIRCULAR-DICHROISM AND FLUORESCENCE SPECTROSCOPY

Circular dichroic spectra in the region 180-260 nm of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis and of four mutants with a Phe residue replaced by Trp, i.e. [F29W]HBsu, [F47W]HBsu, [F50 W]HBsu and [F79W]HBsu, show minor differences only and demonstrate the general similarity of the conformations of these proteins. Fluorescen ce maxima at 315-320 nm and 330-335 nm indicate a more hydrophobic env ironment or a more effective stacking of Trp residues in mutants [F29W ]HBsu and [F50W]HBsu in comparison to [F47W]HBsu and [F79W]HBsu, respe ctively. Unfolding of the mutants in high-ionic-strength buffers by in creasing concentrations of urea results in a red shift of the fluoresc ence emission maxima to about 350 nm; the fluorescence intensities dec rease strongly for [F29W]HBsu and [F50W]HBsu but show a small increase for [F47W]HBsu and [F79W]HBsu. The data suggest complex unfolding pat terns with subtle differences between the single mutants. The circular dichroic spectra in the region 250-320 nm are dominated by the effect s of the Trp residues and signal position-dependent differences in the environment of the Trp residues. The conformations of the mutant prot eins depend on the ionic strength of the buffer and become more stable against unfolding by denaturants or increasing temperatures at higher ionic strength. At low ionic strength a pronounced protein-concentrat ion dependence of the conformation of the mutants is seen.