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首页> 外文期刊>Biochemistry >Reversible dissociation and unfolding of the Escherichia coli aspartate receptor cytoplasmic fragment.
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Reversible dissociation and unfolding of the Escherichia coli aspartate receptor cytoplasmic fragment.

机译：大肠杆菌天冬氨酸受体胞质片段的可逆解离和展开。

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摘要

The thermal denaturation of a 31-kDa soluble fragment derived from the Escherichia coli aspartate receptor cytoplasmic region (c-fragment) was found to be reversible. Denaturation monitored by differential scanning calorimetry (DSC) and circular dichroism (CD) was typically over 90% reversible in pH 7.0 buffer. The wild-type c-fragment exhibited one transition (Tm = 51 degrees C), which was taken as the main denaturation transition. c-Fragments derived from signaling mutants, shown to form oligomers by gel filtration chromatography (GFC), displayed a second low-temperature transition that correlated with the disappearance of the oligomeric form in the GFC traces over the same temperature range. The CD and DSC experiments also indicated that oligomers were more folded than monomers, observations that may provide an explanation for the structural basis of the smooth-swimming signaling state of the receptor. Octyl glucoside (OG), phospholipid (PL), and glycerol were added to characterize factors that contribute to c-fragment stability. At 10 mg/mL OG, the van't Hoff enthalpy of unfolding was reduced ca. 10-fold, although at room temperature the CD spectrum indicated little change in the secondary structure. The van't Hoff enthalpy was not affected by 35% (w/v) glycerol, but the Tm increased by ca. 18 degrees C. Cooperative transitions were detected in buffer containing OG, PL, and glycerol (10 mg/mL, 2 mg/mL, 35%, respectively). The correlation between conditions where cooperative transitions are observed, and where aspartate-modulated receptor signaling has been previously observed, provides an explanation for the inhibition of signaling in OG-containing buffers without glycerol and PL.
机译：发现衍生自大肠杆菌天冬氨酸受体胞质区（c-片段）的31kDa可溶片段的热变性是可逆的。通过差示扫描量热法（DSC）和圆二色性（CD）监测的变性在pH 7.0缓冲液中通常可逆90％以上。野生型c片段表现出一个转变（Tm = 51摄氏度），这被视为主要的变性转变。衍生自信号突变体的c片段，通过凝胶过滤色谱法（GFC）显示形成寡聚物，显示出第二个低温转变，该转变与在同一温度范围内GFC迹线中寡聚形式的消失相关。 CD和DSC实验还表明，寡聚体比单体更易折叠，这一发现可能为受体平滑游动信号状态的结构基础提供了解释。加入辛基葡萄糖苷（OG），磷脂（PL）和甘油来表征有助于c片段稳定性的因子。 OG浓度为10 mg / mL时，展开的范霍夫焓降低。 10倍，尽管在室温下CD光谱表明二级结构几乎没有变化。 Van't Hoff焓不受35％（w / v）甘油的影响，但Tm升高了约5％。 18摄氏度。在含有OG，PL和甘油（分别为10 mg / mL，2 mg / mL，35％）的缓冲液中检测到协同转变。观察到协同转换的条件与先前观察到天冬氨酸调节的受体信号转导的条件之间的相关性，为不含甘油和PL的含OG缓冲液中信号转导的抑制提供了解释。

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