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D. A. Altukhov, Y. K. Agapova, A. V. Vlaskina, D. A. Korzhenevskiy, A. Y. Nikolaeva, A. M. Frank-Kamenetskaya, E. V. Bocharov, T. V. Rakitina
Preparation of the recombinant HU-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
HU-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/13C-HSQC-CT NMR spectra of free HU-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 for the studies of HU-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.
Key words: DNA binding, histone-like HU-proteins, bacterial expression system, NMR.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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