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Cloning, purification and metal binding of the HNH motif from colicin E7

Gyurcsik, Béla and Czene, Anikó and Jankovics, Hajnalka and Jakab-Simon, Noémi I. and Slaska-Kiss, Krystyna and Kiss, Antal and Kele, Zoltán (2013) Cloning, purification and metal binding of the HNH motif from colicin E7. PROTEIN EXPRESSION AND PURIFICATION, 89. pp. 210-218. ISSN 1046-5928

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Abstract

The HNH family of endonucleases is characterized by a betabetaalpha metal-finger structural motif. Colicin E7 is a representative member of this family containing the strictly conserved HNH motif at its C-terminus. Structural and biochemical studies suggested that the HNH motif could contain all the residues necessary for metal ion binding and nuclease activity. In this work a 43 amino acid peptide extending from V534 to K576 of colicin E7 and encompassing the HNH motif was cloned and expressed in Escherichia coli as a ubiquitin fusion protein. The N-terminal fusion tag was cleaved off by a specific protease, and the HNH peptide was purified free of ubiquitin. Circular dichroism, fluorescence and mass spectrometry showed that the zinc-ion binding affinity of the purified HNH peptide was much weaker than that of the intact nuclease domain suggesting that the N-terminal part of the nuclease domain is essential for stabilizing the structure of the HNH motif. The coordination sphere of the metal ion was found to be not fully equipped by the ligand - leaving a free coordination site for the substrate. Neither DNA binding nor DNAse activity of the purified HNH peptide was detected. Comparison of the glutathion-S-transferase-fused N-terminal deletion mutants of the colicin E7 nuclease domain suggested that the presence of the DNA-binding site is still not sufficient for the catalytic activity.

Item Type: Article
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3015 Molecular biology / molekuláris biológia
T Technology / alkalmazott, műszaki tudományok > T2 Technology (General) / műszaki tudományok általában
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 07 Aug 2013 09:57
Last Modified: 07 Aug 2013 09:57
URI: http://real.mtak.hu/id/eprint/6200

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