Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest. Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19, we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

中文翻译：

---

从头开始设计模块化和可调的变构生物传感器。

天然存在的变构蛋白开关已被重新用于开发用于细胞和临床应用的新型生物传感器和报道分子，但是这种开关的数量有限，并且由于每个开关的不同，对其进行工程设计通常具有挑战性。在这里，我们表明可以通过从头设计的蛋白质开关反转信息流来创建非常通用的基于变构蛋白质的生物传感器，其中肽键的结合会触发感兴趣的生物输出。使用广泛适用的设计原则，我们通过设计的锁定蛋白和关键蛋白的结合，将目标蛋白质分析物的结合变构偶联了荧光素酶活性的重建和生物发光读数。由于该传感器完全基于分析物与开关激活的热力学耦合，仅需要一个靶标结合域，从而简化了传感器设计并允许溶液中直接读出。我们通过创建生物传感器来证明该平台的模块化，该传感器几乎没有进行优化，可以灵敏地检测抗凋亡蛋白Bcl-2，hIgG1 Fc结构域，Her2受体和肉毒杆菌神经毒素B，以及心脏肌钙蛋白I和一种抗乙型肝炎病毒（HBV）抗体，可达到检测这些分子临床相关浓度所需的亚纳摩尔级敏感性。鉴于当前需要用于跟踪COVID-19的诊断工具，我们使用该方法设计针对SARS-CoV-2蛋白表位的抗体和SARS-CoV-2 Spike蛋白的受体结合域（RBD）的传感器。后者结合了从头设计的RBD粘合剂，添加RBD时，检测限为15pM，发光增加多达17倍。该平台的模块化和灵敏性应该能够快速构建用于各种分析物的传感器，并强调从头进行蛋白质设计的能力，以创建具有新的有用功能的多状态蛋白质系统。