Nagy Maria, Akoev Vladimir, Zolkiewski Michal
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Arch Biochem Biophys. 2006 Sep 1;453(1):63-9. doi: 10.1016/j.abb.2006.03.004. Epub 2006 Mar 23.
ClpB is a heat-shock protein that reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the family of AAA+ ATPases and forms ring-shaped oligomers: heptamers in the absence of nucleotides and hexamers in the presence of nucleotides. We investigated the thermodynamic stability of ClpB in its monomeric and oligomeric forms. ClpB contains six distinct structural domains: the N-terminal domain involved in substrate binding, two AAA+ ATP-binding modules, each consisting of two domains, and a coiled-coil domain inserted between the AAA+ modules. We produced seven variants of ClpB, each containing a single Trp located in each of the ClpB domains and measured the changes in Trp fluorescence during the equilibrium urea-induced unfolding of ClpB. We found that two structural domains: the small domain of the C-terminal AAA+ module and the coiled-coil domain were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation and/or interactions during formation of the ClpB rings.
ClpB是一种热休克蛋白，它与DnaK伴侣系统协同作用，使聚集的蛋白质重新激活。ClpB属于AAA+ATP酶家族，可形成环状寡聚体：在无核苷酸时形成七聚体，在有核苷酸时形成六聚体。我们研究了ClpB单体和寡聚体形式的热力学稳定性。ClpB包含六个不同的结构域：参与底物结合的N端结构域、两个AAA+ATP结合模块（每个模块由两个结构域组成）以及一个插入在AAA+模块之间的卷曲螺旋结构域。我们制备了七种ClpB变体，每种变体在ClpB的每个结构域中都含有一个单一的色氨酸，并在尿素诱导的ClpB平衡展开过程中测量了色氨酸荧光的变化。我们发现，两个结构域：C端AAA+模块的小结构域和卷曲螺旋结构域在ClpB的寡聚体形式中不稳定，这表明只有那些结构域在ClpB环形成过程中改变了它们的构象和/或相互作用。
