Computational design of protein-based modular small-molecule biosensors

描述(由申请人提供)：感知小分子的能力在探测生物过程、生物工程和医学诊断方面具有重要和广泛的应用。这个R21项目试图证明一种计算设计平台的可行性，以设计基于蛋白质的(因此可遗传编码的)小分子生物传感器。为了实现这一目标，我们将开发和评估方法，将小分子结合位点工程到异二聚体蛋白质-蛋白质界面，使蛋白质-蛋白质相互作用变得依赖于小分子。这两个蛋白质伙伴中的每一个都将连接到分裂报告的一个片段。然后，功能传感器通过报告的互补来检测小分子的存在。以这种方式，传感器输出原则上是模块化的：不同的报告片段可以连接到小分子传感器组件上，并进行检测(通过例如Split-GFP)或激活(Split-酶或基因表达)。我们的新贡献将是开发一种设计方法，该方法使用四步过程将小分子结合位点从连接的蛋白质结构移植到蛋白质-蛋白质界面：(1)给定一个小分子靶标，我们为结合的“基序”(通常是蛋白质侧链的一部分)定义几何约束，该基序在连接的蛋白质单体结构中协调靶标。(2)我们搜索&gt；600异二聚体蛋白质-蛋白质界面(“支架”)，寻找那些可以容纳目标及其结合基序的支架。(3)利用我们最近开发的机器人学方法，我们对靶结合位点周围的支架界面进行了计算改造和设计，以稳定其结合构象中的基序残基。(4)我们预测传感器序列的文库，并仅在小分子存在的情况下使用体外结合分析和在细菌中使用生物传感器输出来测试这些二聚化。模块化小分子诱导二聚反应的计算机辅助设计将是第一次。我们的实验计划试图通过几种方式来改善这个设计驱动的项目中固有的风险：(I)适度的靶结合亲和力旨在使用最近导致成功设计新酶的基序指导方法被预编程到界面中。(2)计算的补充不仅是对最高预测的实验测试，而且是对序列库的实验测试。(3)生物传感器平台促进了对这些文库的测试，预期的输出本身将用于筛选、选择和改进传感器。虽然小分子诱导的蛋白质二聚作用，例如在雷帕霉素系统中，在自然界中存在，并已被重新设计，但这些系统仅限于几个可以感觉到的分子。该项目的成功完成将展示一种平台技术，通过为许多目标提供一条模块化生物传感器的途径，可以极大地扩大小分子传感和驱动的应用。 与公共健康相关：这项研究开发了新技术，以设计由生物成分制成的传感器，从而实时检测活细胞和生物体中的分子。这种新型生物传感器在生物医学研究和生物工程中有许多实际应用，也将通过监测健康和疾病状态的分子来促进我们对基本细胞过程的理解。该项目开发的软件和技术将广泛应用于研究人员，以改进生物传感器的设计。

DESCRIPTION (provided by applicant): The ability to sense small molecules has important and wide-ranging applications for probing biological processes, for biological engineering and for medical diagnostics. This R21 project seeks to demonstrate feasibility of a computational design platform to engineer protein-based (thus genetically encodable) small molecule biosensors. To achieve this goal, we will develop and assess methods to engineer small molecule binding sites into heterodimeric protein-protein interfaces such that the protein-protein interaction becomes dependent on the small molecule. Each of the two protein partners will be linked to a fragment of a split reporter. A functional sensor then detects the presence of the small molecule by complementation of the reporter. In this fashion, the sensor output is in principle modular: different reporter fragments can be attached to the small molecule sensor components and tested for either detection (via for example split-GFP) or actuation (split-enzymes or gene expression). Our novel contribution will be to develop a design approach that uses a four-step process to transplant small molecule binding sites from liganded protein structures into protein-protein interfaces: (1) Given a small molecule target, we define geometric constraints for a binding "motif" (generally parts of protein side chains) that coordinates the target in a liganded protein monomer structure. (2) We search >600 heterodimeric protein-protein interfaces ("scaffolds") for those that could accommodate the target and its binding motif. (3) We computationally remodel and design the scaffold interface around the target-binding site to stabilize the motif residues in their binding conformation, using robotics-inspired approaches we recently developed. (4) We predict libraries of sensor sequences and test these for dimerization only in the presence of the small molecule using in vitro binding assays and in bacteria using the biosensor output. Computer-aided design of modular small-molecule induced dimerization would be a first. Our experimental plan seeks to ameliorate the risk inherent in this design-driven project in several ways: (i) Moderate target binding affinity is intended to be pre-programmed into the interface using a motif- directed approach that has recently led to successful design of new enzymes. (ii) Computation is complemented with experimental testing of not only the top predictions, but libraries of sequences. (iii) Testing these libraries is facilitated by the biosensor platform, where the intended output itself will be used to screen, select and improve sensors. While small-molecule induced protein dimerization, such as in the rapamycin system, exists in nature and has been reengineered, these systems are limited to a few molecules that can be sensed. Successful completion of this project would demonstrate a platform technology that could greatly broaden application of small-molecule sensing and actuation by providing a route to modular biosensors for many targets. PUBLIC HEALTH RELEVANCE: This research develops new technologies to engineer sensors made out of biological components that allow real-time detection of molecules in living cells and organisms. Such new biosensors have many practical applications in biomedical research and biological engineering, and will also help to advance our understanding of fundamental cellular processes by monitoring molecules in health and disease states. The software and technology developed in this project will be widely available to researchers to improve biosensor design.