Robic Srebrenka, Berger James M, Marqusee Susan
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Protein Sci. 2002 Feb;11(2):381-9. doi: 10.1110/ps.38602.
To investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent protein and the remaining parts of the other. Both chimeras form active, well-folded RNases H. Stability curves, based on CD-monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (DeltaCp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low DeltaCp of both the parent thermophilic RNase H and the chimera with the thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the thermophilic core resembles a true thermophilic protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H.
为了研究折叠核心对核糖核酸酶H热力学稳定性的贡献，我们通过合理设计构建了两种嵌合体，它们由嗜热核糖核酸酶H和嗜温核糖核酸酶H的部分片段组成。每种嵌合体都结合了一种亲本蛋白的折叠核心和另一种亲本蛋白的其余部分。两种嵌合体均形成了具有活性且折叠良好的核糖核酸酶H。基于圆二色光谱监测的化学变性得到的稳定性曲线表明，具有嗜热核心的嵌合体比具有嗜温核心的嵌合体更稳定，热变性中点更高，且去折叠时的热容变化（ΔCp）更低。亲本嗜热核糖核酸酶H和具有嗜热核心的嵌合体的ΔCp较低的一个可能解释是变性状态下的残余结构。基于所研究的参数，具有嗜热核心的嵌合体类似于真正的嗜热蛋白。我们的结果表明，折叠核心在赋予核糖核酸酶H热力学参数方面起着至关重要的作用。
