Merz Tobias, Wetzel Svava K, Firbank Susan, Plückthun Andreas, Grütter Markus G, Mittl Peer R E
Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
J Mol Biol. 2008 Feb 8;376(1):232-40. doi: 10.1016/j.jmb.2007.11.047. Epub 2007 Nov 22.
Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 A resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI(3)C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.
全共识设计的锚蛋白重复序列蛋白（DARPins）已得到表征，其中先前描述的DARPin文库的随机位置已被固定。它们表现出异常高的热力学稳定性，即使与共识DARPin文库的成员相比也是如此，与天然存在的锚蛋白重复序列蛋白相比更是如此。我们确定了一个全共识DARPin的晶体结构，其分辨率为2.05埃，包含一个N端封端重复序列、三个相同的内部重复序列和一个C端封端重复序列，并将其结构与相关的DARPin文库成员E3_5和E3_19的结构进行了比较。这种结构比较表明，主要是表面上的盐桥以几乎晶体般的规则排列成网络，增加了全共识NI(3)C DARPin的热稳定性，使其能够耐受煮沸。在晶体结构中，三个硫酸根离子补充了这个网络。在盐酸胍中进行的热变性实验直接表明硫酸根结合对稳定性有贡献，为表面暴露的静电相互作用和规则电荷网络的稳定作用提供了进一步的证据。基于序列统计，选择了DARPin文库中随机化残基位置处的带电残基，这表明电荷相互作用网络是这个蛋白质家族隐藏的设计特征。因此，锚蛋白可以利用嗜热生物蛋白质的设计原则，达到至少相似的稳定性。
