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Single chain dimers of MASH-1 bind DNA with enhanced affinity

Sieber, M. and Allemann, Rudolf Konrad 1998. Single chain dimers of MASH-1 bind DNA with enhanced affinity. Nucleic Acids Research 26 (6) , pp. 1408-1413. 10.1093/nar/26.6.1408

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Abstract

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of ∼55 Å, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no ‘cross-folding’ of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly α-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased ∼10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from −0.14 to −0.57 kcal/mol.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Cardiff Catalysis Institute (CCI)
Subjects: Q Science > QD Chemistry
Publisher: Oxford University Press
ISSN: 0305-1048
Last Modified: 04 Jun 2017 02:53
URI: http://orca.cf.ac.uk/id/eprint/13463

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