Biophysical and structural property of the putative DNA-binding protein, BldB, from Streptomyces lividans.

ABSTRACT

The bldB gene from Streptomyces lividans was cloned, and its product was overexpressed in Escherichia coli using a T7 expression system. Gel mobility shift assays showed that the BldB protein was functionally expressed in the E. coli system and may negatively regulate its own expression. The comparative analyses by mass spectrometry, Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analytical ultracentrifuge established that BldB is a dimeric protein with 24 kDa molecular mass, of which monomers do not covalently interact with each other. Gel filtration result implied that the protein shape would not be globular. More detailed structural investigations by CD and NMR spectroscopy confirmed that the majority of the BldB structure is not only disordered but also highly flexible. The highly reversible, but hardly cooperative, property of the thermal denaturation also supported the idea that the protein structure is not compact. However, the existence of a structural nucleus, of which the ordered conformation remains stabilized even at more than 80 degrees C, was evidenced. The overall structure and the thermal stability of BldB were sensitive to pH, suggesting a proton-induced conformation change. Altogether, the results provide the first detailed characterization on the biophysical and structural property of the putative DNA-binding protein, BldB.