K-unne A G, Sieber M, Meierhans D, Allemann R K
Department of Chemistry, ETH-Zurich, Universitätstrasse 16, CH-8092 Zurich, Switzerland.
Biochemistry. 1998 Mar 24;37(12):4217-23. doi: 10.1021/bi9725374.
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".
MASH-1是转录因子基本螺旋-环-螺旋（BHLH）家族的成员，可促进定向神经元前体细胞的分化。我们通过等温滴定量热法测定了MASH-1的BHLH结构域（MASH-BHLH）与DNA结合反应的热力学参数，发现结合反应的特异性相当低。在27摄氏度时，对于含E-box的寡核苷酸，结合的缔合常数为5.13（±0.51）×10⁸ M⁻¹，而对于异源DNA序列，缔合常数为5.14（±1.93）×10⁷ M⁻¹。反应焓和反应熵强烈依赖于温度，但反应自由能几乎与温度无关。在整个生理温度范围内，缔合反应是由焓驱动的，其特征是有很大的负热容变化。在pH 6时未观察到蛋白质和/或DNA质子化状态的变化。在实验误差范围内，该反应在pH 6至8之间与pH无关。对结合反应熵变的剖析表明，结合与每个蛋白质亚基约25个氨基酸的局部蛋白质折叠相关。游离的和与DNA结合的MASH-BHLH的圆二色光谱显示，复合物形成时会形成包含约25个氨基酸的额外α-螺旋结构。因此，虽然在DNA复合物中碱性区域呈α-螺旋构象，但在游离的MASH-BHLH中，即使在蛋白质主要为二聚体的浓度下，它也基本处于未折叠状态。因此，MASH-1与DNA之间的缔合是“诱导契合”的一个例子。
