Thermodynamics of DNA binding of MM17, a 'single chain dimer' of transcription factor MASH-1.
MASH-1, a member of the basic helix-loop-helix (bHLH) family of transcriptional regulators, is a central factor for the regulation of the differentiation of committed neuronal precursor cells of the peripheral nervous system. We have previously produced MM17, a single chain version of this dimeric protein, by linking the C-terminal end of the first subunit to the N-terminal residue of the second subunit through a flexible peptide linker. We have now determined by isothermal titration calorimetry the thermodynamic parameters characterising the DNA binding reactions of MM17. The DNA binding specificity was relatively low and comparable to that observed for wild-type MASH bHLH. At 32 degrees C and pH 7, the concentration of MM17 at which 50% DNA binding occurred was determined as 22.8 and 152 nM for binding to MCK-S and the heterologous SP-1, respectively. Similarly to MASH bHLH the free energy of the association was only slightly temperature dependent, while both the entropy and the enthalpy change were strong functions of temperature. The free energy of DNA binding was independent of the pH for the pH range between 6 and 8. Dissection of the entropy change of the association reaction suggested that the two basic domains and the linker region between the subunits underwent a folding transition from a mainly unfolded to a predominantly ordered conformation. Therefore, like wild-type MASH bHLH, the DNA binding reaction of MM17 follows an induced fit mechanism.
Sieber
M
M
School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Allemann
R K
RK
D000595:Amino Acid Sequence; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002151:Calorimetry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D019277:Entropy; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; C065500:ASCL1 protein, human