P61A mutation in the factor for inversion stimulation results in a thermostable dimeric intermediate.
P61A mutation in the factor for inversion stimulation results in a thermostable dimeric intermediate.
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反转刺激因子中的 P61A 突变会产生热稳定的二聚体中间体。
DOI：
10.1021/bi050640k
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
W. Colón
中科院分区：
文献类型：
--
作者：
Derrick W. Meinhold;Sarah A. Boswell;W. Colón
倒位刺激因子（FIS）是一种在肠道细菌中发现的同源二聚体DNA结合蛋白。FIS由98个残基组成，并自组装成包含柔性且大部分无序的N-末端，随后是四个α-螺旋的包封二聚体。脯氨酸61在FIS同源物中是100%保守的，位于螺旋B的中心，并且其对丙氨酸（P61 A）的取代先前显示导致蛋白质的不均匀稳定，导致在尿素变性平衡研究中出现边缘填充的二聚体中间体。在这里，我们表明，与WT FIS相反，P61A FIS的热变性是不完全的，并产生了一个过渡曲线，这是独立的FIS浓度，表明在90 ℃的二聚体中间体的存在。在尿素的存在下，P61A FIS的热变性变得依赖于浓度，与二聚体中间体的变性一致。通过95 ℃下的戊二醛交联实验进一步证实了P61A FIS的热稳定二聚体中间体的存在。尿素变性实验在90摄氏度揭示了一个合作的转变，表明二聚体中间体的P61A FIS有一个溶剂保护的疏水核心。与WT蛋白不同，P61A FIS被发现对低pH变性具有抗性，但其在pH 3.5下的热变性揭示了双相转变，提供了关于二聚体中间体结构的线索。从功能的角度来看，这是合理的，在FIS中的脯氨酸61的完全保守可能会限制这种高度调节的转录因子的稳定性和蛋白水解抗性。
The factor for inversion stimulation (FIS) is a homodimeric DNA-binding protein found in enteric bacteria. FIS consists of 98 residues and self-assembles into an entwined dimer containing a flexible and mostly disordered N-terminus followed by four alpha-helices. Proline 61, which is 100% conserved in FIS homologues, is located at the center of helix B, and its substitution for alanine (P61A) was previously shown to result in nonuniform stabilization of the protein, leading to the appearance of a marginally populated dimeric intermediate in urea denaturation equilibrium studies. Here we show that, in contrast to WT FIS, the thermal denaturation of P61A FIS was incomplete and yielded a transition curve that was independent of FIS concentration, suggesting the presence of a dimeric intermediate at 90 degrees C. In the presence of urea, the thermal denaturation of P61A FIS became concentration dependent, consistent with the denaturation of the dimeric intermediate. The existence of a thermostable dimeric intermediate of P61A FIS was further confirmed by glutaraldehyde cross-linking experiments at 95 degrees C. Urea denaturation experiments at 90 degrees C revealed a cooperative transition, indicating that the dimeric intermediate of P61A FIS has a solvent-protected hydrophobic core. P61A FIS, unlike the WT protein, was found to be resistant to denaturation by low pH, but its thermal denaturation at pH 3.5 revealed a biphasic transition, providing clues about the structure of the dimeric intermediate. From a functional perspective, it is plausible that the full conservation of proline 61 in FIS may serve to limit the stability and proteolytic resistance of this highly regulated transcription factor.
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嗜热蛋白质的物理基础：丰富的极性相互作用和降低的展开热容量的联系。
DOI：
10.1016/s0006-3495(02)75316-2
发表时间：
2002
期刊：
Biophysical journal.
影响因子：
--
作者：
Zhou,Huan-Xiang
通讯作者：
Zhou,Huan-Xiang
酪氨酸残基对反转刺激因子的结构和光谱特性的不同贡献。
DOI：
10.1021/bi035441k
发表时间：
2004
期刊：
Biochemistry.
影响因子：
--
作者：
Boswell,Sarah;Mathew,John;Beach,Michael;Osuna,Robert;Colon,Wilfredo
通讯作者：
Colon,Wilfredo
影响因子：
56.9
作者：
HARBURY, PB;ZHANG, T;ALBER, T
通讯作者：
ALBER, T
影响因子：
3.4
作者：
M. Torrez;Michael Schultehenrich;D. Livesay
通讯作者：
M. Torrez;Michael Schultehenrich;D. Livesay
影响因子：
2.9
作者：
LUMB, KJ;KIM, PS
通讯作者：
KIM, PS