CAREER: Defining structural principles for the engineering and evolution of allostery

蛋白质是纳米级分子，其组成和形状由基因指定。在细胞内，它们催化化学反应，进行机械工作，并组装成更大的结构。许多蛋白质的集体作用驱动细胞生长、分裂和运动。蛋白质通常由外部信号控制-例如，向细胞环境中添加营养物质可能会通过与蛋白质结合并改变其形状或动力学来打开或关闭蛋白质。这种变构控制为细胞感知和响应环境提供了基本机制，并且是细胞内通信的构建块。完全理解变构控制如何工作，以及它如何在蛋白质的遗传序列中编码，将使生物学家能够设计出对人工信号做出反应的蛋白质。 在这个项目中，PI的研究团队将使用计算和实验来了解蛋白质中的变构调节是如何优化的，定义区分变构表面的物理特性，并构建一组合成的变构开关，从而能够用光来控制细胞生长速率。这项工作将建立一个实用的，通用的工具包工程变构调节，并提供基本的见解如何自然变构调节可能演变。该计划将培养研究生、本科生和生物方向的高中生，提高他们在基础编程和研究实验方面的技能，为STEM人才的发展做出贡献。虽然变构是蛋白质的基本和共同特征，但蛋白质序列和结构如何编码变构仍不清楚。例如，不知道影响变构的突变的模式和数量是稀疏还是丰富，或者对调节具有最大影响的突变是定位于变构位点还是分布在整个结构中。该项目将通过使用深度突变扫描来表征可以影响合成变构开关中的变构调节的完整突变集来解决这些基本问题。这项研究还将使用NMR光谱学的新方法，以更好地了解蛋白质与水的相互作用如何确定（和识别）变构表面。该假说是变构表面以减慢的水合动力学为标志。如果是这样的话，这将表明变构位点是新蛋白质和配体结合相互作用进化的熵优选位点。此外，这将为发现具有变构作用的药物开辟新的途径。最后，通过这些突变和结构研究获得的知识将被应用于产生一系列宽动态范围的变构开关，用于基于光的细胞生长控制。这些开关将为动态调节细胞生长提供实用的工具包，在细菌群落动力学研究、真核细胞增殖和工程活生物治疗方面具有潜在的应用。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Proteins are nanometer-scale molecules whose composition and shape are specified by genes. Inside the cell, they catalyze chemical reactions, perform mechanical work, and assemble into larger structures. The collective action of very many proteins drives cell growth, division, and movement. Proteins are often controlled by external signals – for example, adding a nutrient to the cellular environment might turn a protein “on” or “off” by binding to the protein and altering its shape or dynamics. Such allosteric control provides a basic mechanism for cells to sense and respond to the environment and is a building block for intracellular communication. A complete understanding of how allosteric control works, and how it is encoded in the genetic sequence of proteins, would allow biologists to engineer proteins that respond to artificial cues. In this project, the PI’s research team will use computation and experiment to understand how allosteric regulation in a protein is optimized, define physical properties distinguishing allosteric surfaces, and construct a set of synthetic allosteric switches that enable control of cell growth rate with light. This work will establish a practical, general toolkit for engineering allosteric regulation, and provide fundamental insights into how natural allosteric regulation might evolve. This proposal will train graduate, undergraduate, and bio-oriented high school students to improve their skills in basic programming and research experimentation to contribute STEM workforce development.Though allostery is a fundamental and common feature of proteins, how it is encoded by protein sequence and structure remains unclear. For example, it is unknown if the pattern and number of mutations influencing allostery is sparse or abundant, or if the mutations with the biggest effect on regulation are localized to the allosteric site or distributed throughout the structure. This project will address these fundamental questions by using deep mutational scanning to characterize the complete set of mutations than can influence allosteric regulation in a synthetic allosteric switch. This research will also use new approaches in NMR spectroscopy to better understand how protein interactions with water might determine (and identify) allosteric surfaces. The hypothesis is that allosteric surfaces are marked by slowed hydration dynamics. If so, this would suggest that allosteric sites are entropically preferred sites for the evolution of new protein and ligand binding interactions. Moreover, this would open new avenues for the discovery of allosterically acting pharmaceuticals. Finally, the knowledge gained through these mutational and structural studies will be applied to generate a series of broad dynamic range allosteric switches for the light-based control of cell growth. These switches will provide a practical toolkit for dynamically modulating cell growth, with potential applications in the study of bacterial community dynamics, eukaryotic cell proliferation, and engineering live biotherapeutics.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.