Thermodynamics of the DNA binding reaction of transcription factor MASH-1.
MASH-1, a member of the basic helix-loop-helix (BHLH) family of transcription factors, promotes the differentiation of committed neuronal precursor cells. We have determined the thermodynamic parameters of the DNA binding reaction of the BHLH domain of MASH-1 (MASH-BHLH) by isothermal titration calorimetry and found that the specificity of the binding reaction was rather low. At 27 degrees C, the association constant for binding was 5.13 (+/-0.51) x 10(8) M-1 for an E-box containing oligonucleotide, while for a heterologous DNA sequence it was 5.14 (+/-1.93) x 10(7) M-1. The reaction enthalpy and the reaction entropy were strongly dependent on the temperature, but the reaction free energy was almost independent of temperature. The association reaction was enthalpically driven throughout the physiological temperature range and characterized by a large negative heat capacity change. No change in the protonation state of the protein and/or the DNA was observed at pH 6. Within experimental error, the reaction was independent of pH between pH 6 and 8. Dissection of the entropy change of the binding reaction indicated that binding was coupled to local protein folding of approximately 25 amino acids per protein subunit. The circular dichroism spectra of free and DNA-bound MASH-BHLH revealed the formation of additional alpha-helical structure comprising approximately 25 amino acids upon complex formation. Therefore, while the basic region was in an alpha-helical conformation in the DNA complex, in free MASH-BHLH it was substantially unfolded even at concentrations where the protein is mainly dimeric. The association between MASH-1 and DNA is therefore an example of "induced fit".
K-unne
A G
AG
Department of Chemistry, ETH-Zurich, Universitätstrasse 16, CH-8092 Zurich, Switzerland.
Sieber
M
M
Meierhans
D
D
Allemann
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D002151:Calorimetry; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C499318:Ascl1 protein, rat