Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Biochemistry. 2023 Jan 17;62(2):318-329. doi: 10.1021/acs.biochem.2c00083. Epub 2022 Jun 3.
High protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap.
蛋白质稳定性高是将蛋白质用作治疗药物、诊断试剂和基础研究的重要特性。我们之前曾采用共识设计来工程化优化的角蛋白重复蛋白（ArmRPs），以实现线性表位的序列特异性识别，具有模块化的结合模式。这些设计的 ArmRPs（dArmRPs）具有高稳定性，由 M 型内部重复组成，两端为 N 端和 C 端盖帽重复，保护疏水区免受溶剂暴露。虽然设计的 ArmRPs 的整体稳定性非常高，但随后的生化和生物物理实验表明，N 端盖帽重复在一小部分时间内呈现部分展开、可溶剂的构象，使其容易受到蛋白水解和聚集的影响。为了解决这个问题，我们使用 Rosetta 软件从 M 型内部重复开始设计新的 N 帽。通过圆二色性和核磁共振波谱实验验证了计算优化模型的卓越稳定性。包含新型 N 帽的 dArmRP 的晶体结构表明，增强的稳定性与该 N 帽在 dArmRP 疏水区上的改进堆积相关。氢交换实验进一步表明，N 帽的局部展开水平降低了几个数量级，从而增加了对蛋白水解的抗性和减弱了聚集。作为新型 N 帽的首次应用，我们确定了具有四个内部重复的 dArmRP 的溶液结构，这在以前由于原始 N 帽的不稳定性而受到阻碍。
