Ha Jeung-Hoi, Butler James S, Mitrea Diana M, Loh Stewart N
Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, 750 E. Adams St, Syracuse, NY 13210, USA.
J Mol Biol. 2006 Apr 7;357(4):1058-62. doi: 10.1016/j.jmb.2006.01.073. Epub 2006 Feb 6.
A regulatory mechanism is introduced whereupon the catalytic activity of a given enzyme is controlled by ligand binding to a receptor domain of choice. A small enzyme (barnase) and a ligand-binding polypeptide (GCN4) are fused so that a simple topological constraint prevents them from existing simultaneously in their folded states. The two domains consequently engage in a thermodynamic tug-of-war in which the more stable domain forces the less stable domain to unfold. In the absence of ligand, the barnase domain is more stable and is therefore folded and active; the GCN4 domain is substantially unstructured. DNA binding induces folding of GCN4, forcibly unfolding and inactivating the barnase domain. Barnase-GCN4 is thus a "natively unfolded" protein that uses ligand binding to switch between partially folded forms. The key characteristics of each parent protein (catalytic efficiency of barnase, DNA binding affinity and sequence specificity of GCN4) are retained in the chimera. Barnase-GCN4 thus defines a modular approach for assembling enzymes with novel sensor capabilities from a variety of catalytic and ligand binding domains.
引入了一种调节机制，据此特定酶的催化活性由配体与所选受体结构域的结合来控制。一种小酶（芽孢杆菌RNA酶）和一种配体结合多肽（GCN4）融合在一起，从而一种简单的拓扑限制阻止它们同时以折叠状态存在。因此，这两个结构域进行了一场热力学拔河比赛，其中更稳定的结构域迫使较不稳定的结构域展开。在没有配体的情况下，芽孢杆菌RNA酶结构域更稳定，因此折叠且有活性；GCN4结构域基本上是无结构的。DNA结合诱导GCN4折叠，强行展开并使芽孢杆菌RNA酶结构域失活。因此，芽孢杆菌RNA酶-GCN4是一种“天然未折叠”的蛋白质，它利用配体结合在部分折叠形式之间切换。每个亲本蛋白质的关键特性（芽孢杆菌RNA酶的催化效率、GCN4的DNA结合亲和力和序列特异性）在嵌合体中得以保留。因此，芽孢杆菌RNA酶-GCN4定义了一种模块化方法，用于从各种催化和配体结合结构域组装具有新型传感能力的酶。
