Characterization of HS29K nucleoid associated DNA binding protein with Mn +2 ‐ SOD activity from Halophilic Archaeon Halobacterium salinarium strain H3

Abstract

An abundant 29 kDa protein is associated with nucleoid fractions from the haloarchaeon, Halobacterium salinarium. The protein was purified and its properties were studied by fluorescence and circular dichroism spectroscopy. The protein undergoes salt dependent conformational change. The DNA binding properties of the protein were studied by mobility shift assays, fluorescence titrations, and circular dichroism. The binding affinity of the protein to both single and double stranded DNA was stronger at high salt concentrations compared to single stranded RNA as indicated by the extent of fluorescence quenching. Gel mobility shift assays also showed stronger binding of the protein to DNA at high salt concentration. CD spectroscopy indicated that the binding of the protein to DNA resulted in structural change to A form like DNA. The 29 kDa DNA binding protein was identified as Manganese Superoxide Dismutase (Mn-SOD) by MALDI-TOFF analysis. Mass spectra of the intact protein gave Mr. of 22,190. In gel activity assay of the protein showed Mn-SOD activity. Fluorescence titrations of the protein with manganese ions resulted in quenching of intrinsic fluorescence indicating conformational change in the protein as a result of binding of manganese ions. Mn-SOD associated with nucleoid in Halobacterium could be a multifunctional protein involved in DNA metabolism, regulation of transcription and in protection of DNA against oxidative damage.