A synthetic gene encoding the histone‐like DNA‐binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild‐type protein. N‐terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli.. Site‐directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA‐binding parameters by fluorescence spectroscopy. According to gel‐retardation experiments, the mutant protein [Phe47 → Trp]HBsu shows identical DNA binding to wild‐type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47 → Trp]HBsu binds double‐stranded DNA with a dissociation constant in the micromolar range. Computer‐assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47 → Trp]HBsu appears to contact three or four base pairs of DNA. These results are in pritial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.

HBsu. The data suggest complex unfolding patterns with subtle differences between the single mutants. The circular dichroic spectra in the region 250-320 nm are dominated by the effects of the Trp residues and signal position-dependent differences in the environment of the Trp residues. The conformations of the mutant proteins 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-concentration dependence of the conformation of the mutants is seen.Small DNA-binding histone-like bacterial proteins merit interest because of their functional importance. As shown by extensive studies on the HU protein from Escherichia coli, members of this class are involved in processes such as replication, transcription and transposition [l]. These proteins share some properties with eukaryotic histones, e.g. small size, basic isoelectric point and an amino acid composition rich in lysine residues. Furthermore they are capable of wrapping DNA into bead-like structures [l]. They bind non-specifically to double-and single-stranded DNA and RNA [2-41. Most of the proteins form homodimers, but for proteins from E. coli [ 5 ] and from Salmonella typhiinuriunz [6] heterodimers have been described. The primary structures of several proteins are known and are highly conserved [7]. is known at a resolution of 0.21 nm, and models of its complexes with DNA were suggested [8, 91. Of the 90 amino acids of a HBst monomer 12 are replaced by others in the sequence of HBsu from Bacillus subtilis. The HBsu monomer contains 92 amino acids, two more than HBst, and has a molecular mass of 9882Da. The structure of HBsu undergoes salt-dependent and protein-concentration-dependent changes in solution [lo]. Synthesis and cloning of a gene coding for HBsu protein [ l l ] and cloning and mapping of the B. subtilis chromosomal hbsu gene [12] were described recently. This permitted the introduction of fluorophores by site-directed mutagenesis and the determination of DNA-binding parameters of HBsu [13].From CD studies in the far-ultraviolet region of the spectra general information on the folding of the polypeptide chain of a protein can be derived, whereas CD spectra in the near-ultraviolet region more specifically reflect local conformational properties connected with aromatic amino acid side chains [14], especially Trp residues. Fluorescence spectra also contain information depending on the electronic states of aromatic residues and thus permit very sensitive monitoring of conformational changes of a protein in the vicinity of these residues.Here we describe a comparative analysis of wild-type HBsu and its four mutant proteins [F29W]HBsu, [F47W]HBsu, [FSOWIHBsu and [F79W]HBsu by circular dichroism and fluorescence measurements. In each of these