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

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• 作者：D. A. Altukhov ; Y. K. Agapova ; A. V. Vlaskina…
• 关键词：DNA binding ; histone ; like НU ; proteins ; bacterial expression system ; NMR
• 刊名：Moscow University Chemistry Bulletin
• 出版年：2016
• 出版时间：July 2016
• 年：2016
• 卷：71
• 期：4
• 页码：221-226
• 全文大小：746 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Chemistry
  Russian Library of Science
  
• 出版者：Allerton Press, Inc. distributed exclusively by Springer Science+Business Media LLC
• ISSN：1935-0260
• 卷排序：71

文摘

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.