Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor.
Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor.
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DOI：
10.1021/bi034416b
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发表时间：
2003-08
期刊：
影响因子：
2.9
通讯作者：
A. Gelinas;J. Toth;Kelley A. Bethoney;K. Langsetmo;W. Stafford;C. Harrison
中科院分区：
文献类型：
--
作者：
A. Gelinas;J. Toth;Kelley A. Bethoney;K. Langsetmo;W. Stafford;C. Harrison
GrpE是大肠杆菌分子伴侣DnaK的核苷酸交换因子，DnaK是Hsp 70的细菌同系物。在细菌热休克反应的温度范围内，GrpE的长螺旋经历螺旋到卷曲的转变，并且GrpE就其核苷酸交换功能而言表现出非阿伦尼乌斯行为。据推测，GrpE作为一种热敏元件，其长螺旋的解旋可能与E. coliGrpE降低其作为核苷酸交换因子的活性。反过来，有人提出GrpE活性的温度依赖性下调可能会增加DnaK在较高温度下结合其底物的时间。热力学和流体动力学技术的组合，与荧光素酶重折叠试验，被用来表征的分子机制，其中的长螺旋的GrpE是通过Phe 86和Arg 183之间的分子内接触与β-结构域共价连接。这些“热敏”长螺旋被认为是必要的，作为一个核苷酸交换因子在荧光素酶重折叠测定的全部活动。β结构域和GrpE长螺旋中的点突变使β结构域不稳定。长螺旋中的工程化二硫键交替稳定长螺旋和四螺旋束。这使得先前报道的75摄氏度的热转变中看到的过剩热容功能，通过差示扫描量热法监测，以进一步表征。所观察到的热转变代表了四螺旋束和β结构域的展开。这两个域的热转变是叠加的，但不相互关联。
GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70. In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function. It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E. coli GrpE reduces its activity as a nucleotide exchange factor. In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures. A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183. These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay. Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains. Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle. This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized. The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains. The thermal transitions for these two domains are superimposed but are not thermodynamically linked.