Johnson C R, Morin P E, Arrowsmith C H, Freire E
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry. 1995 Apr 25;34(16):5309-16. doi: 10.1021/bi00016a002.
The structural stability of an amino acid fragment containing the oligomerization domain (residues 303-366) of the tumor suppressor p53 has been studied using high-precision differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD). Previous NMR solution structural determinations have revealed that the fragment forms a symmetric 29.8 kDa tetramer composed of a dimer of dimers (p53tet) [Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D., & Arrowsmith, C. H. (1994) Nature Struct. Biol. 1, 877-890]. Thermal unfolding of the tetramer is reversible and can be described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers (N4<==>4U). According to the DSC and CD data, the population of intermediate species consisting of folded monomers or dimers is insignificant, indicating that isolated dimeric or monomeric structures have a much lower stability than the dimer and do not become populated during thermal denaturation under the conditions studied. The transition temperature of unfolding is found to be highly dependent on protein concentration and to follow the expected behavior for a tetramer that dissociates upon unfolding. Experiments conducted at pH 4.0 in 25 mM sodium acetate at a tetramer concentration of 145.8 microM have a transition temperature (Tm) of 75.3 degrees C while at 0.5 microM the value drops to 39.2 degrees C. The enthalpy change of unfolding at 60 degrees C is 26 kcal (mol of monomer)-1 with a heat capacity change of 387 cal (K.mol of monomer)-1. The stability of p53tet is dependent on pH and salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
利用高精度差示扫描量热法（DSC）和圆二色光谱法（CD），对含有肿瘤抑制蛋白p53寡聚化结构域（残基303 - 366）的氨基酸片段的结构稳定性进行了研究。先前的核磁共振溶液结构测定表明，该片段形成了由二聚体的二聚体组成的对称29.8 kDa四聚体（p53tet）[Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D., & Arrowsmith, C. H. (1994) Nature Struct. Biol. 1, 877 - 890]。四聚体的热解折叠是可逆的，可描述为一种两态转变，其中折叠的四聚体直接转变为未折叠的单体（N4<==>4U）。根据DSC和CD数据，由折叠的单体或二聚体组成的中间物种的数量微不足道，这表明分离的二聚体或单体结构的稳定性远低于二聚体，并且在所研究的条件下热变性过程中不会形成。发现解折叠的转变温度高度依赖于蛋白质浓度，并遵循解折叠时解离的四聚体的预期行为。在pH 4.0、25 mM醋酸钠、四聚体浓度为145.8 microM的条件下进行的实验，转变温度（Tm）为75.3℃，而在0.5 microM时，该值降至39.2℃。60℃时解折叠的焓变为26 kcal（每摩尔单体）-1，热容变为387 cal（K·每摩尔单体）-1。p53tet的稳定性取决于pH和盐浓度。（摘要截断于250字）
