The HU protein from Thermotoga maritima: Recombinant expression, purification and physicochemical characterization of an extremely hyperthermophilic DNA-binding protein

The histone-like protein TmHU from the hyperthermophilic eubacterium Thermo toga maritima was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and cation exchange chromato graphy. CD spectroscopical studies with secondary structure analysis as wel l as comparative modeling demonstrate that the dimeric TmHU has a tertiary structure similar to other homologous HU proteins. The T-m of the protein w as determined to be 96 degrees C, and thermal unfolding is nearly completel y reversible. Surface plasmon resonance measurements for TmHU show that the protein binds to DNA in a highly cooperative manner, with a K-D of 73 nM a nd a Kill coefficient of 7.6 for a 56 bp DNA fragment. It is demonstrated t hat TmHU is capable to increase the melting point of a synthetic, double-st randed DNA (poly[d(A-T)]) by 47 degrees C, thus suggesting that DNA stabili zation may be a major function of this protein in hyperthermophiles. The si gnificant in vitro protection of double-helical DNA may be useful for biote chnological applications. (C) 1999 Academic Press.