Preparation of the recombinant НU-proteins from S. melliferum and M. gallisepticum and of their complexes with DS-DNA for structural NMR experiments

Abstract

We created a high-performance system for expression of ¹³C, ¹⁵N-double-labeled recombinant НU-proteins from mycoplasms S. melliferum (HUSpm) and M. gallisepticum (HUMgal), and a purification protocol that yields protein samples suitable for structure studies by high-resolution NMR. Using these proteins we optimized the conditions of NMR experiments and obtained two-dimensional heteronuclear ¹H/¹⁵N-HSQC and ¹H/¹³С-HSQC-CT NMR spectra of free НU-proteins (HUSpm and HUMGal), and 1H/15N-HSQC spectrum of a complex between HUSpm and a double-stranded DNA (ds-DNA). The techniques we created are applicable in the studies of НU-proteins from other organisms. They allow analyzing structure and dynamics of free proteins and of their conformational changes induced by binding of various ds-DNAs or of DNA binding inhibitors.