Single chain dimers of MASH-1 bind DNA with enhanced affinity.
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 approximately 55 A, 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 alpha-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 approximately 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.
Sieber
M
M
Laboratory for Organic Chemistry, Department of Chemistry, ETH-Zürich, Universitätstrasse 16, CH-8092 Zurich, Switzerland.
Allemann
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C065500:ASCL1 protein, human