Computational design of proteins and protein functions

项目摘要/摘要 我们的长期目标是推进计算蛋白质设计，以设计新的生物功能和 分子/细胞工程战略，以揭示生物调节原理。这项建议结合了 我们在这些领域的两笔NIGMS赠款。 在我们的计算蛋白质设计工作中，我们有计算机工程的蛋白质来感知和 对细胞中新的小分子信号做出反应，这是一种在代谢工程中具有重要应用的能力， 诊断、生物修复和探测基本的细胞过程。我们也有先进的方法来 完全从头开始设计形状精确可调的蛋白质。拟议的工作建立在我们新方法的基础上 为了解决一个中心悬而未决的挑战，同时设计从头蛋白的几何结构和用户- 定义的功能部位被放置在其中，原子精度针对功能进行了优化。这项工作应该大有作为 拓展可以设计的新功能的空间。我们计划将从头设计的蛋白质整合到 可以控制生物行为以响应新信号的模块化系统。 我们对天然蛋白质功能的研究试图了解中央调节蛋白质是如何在 相互连接的蜂窝网络，以及这些网络在突变等扰动下是如何改变的。 我们研究了由相反的调节器控制的两态开关(GTP酶)，因为这个基序在 生物学。通过系统地诱变GTPase Gsp1并整合系统的测量 Scale(遗传相互作图)结合生物物理学，我们发现了以前未知的变构位点 GTP酶在其功能中起中心作用。此外，我们的发现还提出了一种新的模型，即多效性GTP酶Gsp1 以不同的方式调节不同的细胞功能。在这里，我们将以这些结果为基础来研究其机制 Gsp1中的变构作用，并评估其在其他GTP酶中的普遍性，这对理解机制有意义 用于疾病突变和调节子的开发。我们还计划测试我们的GTP酶调控模型 通过确定微调扰动对GTP酶调节的定量细胞后果。未来 方向包括将这些扰动测量扩展到其他中枢生物开关。这个 未发现的细胞控制原理可以指导细胞工程，并与计算相结合 设计的新功能可能最终会导致新的方法来对抗疾病中的不当调控。

PROJECT SUMMARY/ABSTRACT Our long-term goals are to advance computational protein design to engineer new biological functions and molecular/cellular engineering strategies to uncover principles of biological regulation. This proposal combines our two NIGMS grants in these areas. In our work on computational protein design, we have computationally engineered proteins that sense and respond to new small molecule signals in cells, a capability with important applications in metabolic engineering, diagnostics, bioremediation, and probing fundamental cellular processes. We have also advanced methods to design proteins with precisely tunable shapes entirely de novo. The proposed work builds on our new methods to address a central unsolved challenge, to simultaneously design the geometries of de novo proteins and user- defined functional sites placed into them with atomic accuracy optimized for function. This work should greatly expand the space of new functions that can be designed. We plan to integrate de novo designed proteins into modular systems that can control biological behavior in response to new signals. Our work on natural protein functions seeks to understand how central regulatory proteins operate in interconnected cellular networks, and how these networks are altered upon perturbations such as mutations. We studied a two-state switch (a GTPase) controlled by opposing regulators because this motif is prevalent in biology. Through systematic mutagenesis of the GTPase Gsp1 and integrating measurements at the systems scale (genetic interaction mapping) with biophysics, we uncovered previously unknown allosteric sites on the GTPase central to its function. Our findings moreover suggest a new model how the pleiotropic GTPase Gsp1 differentially regulates distinct cellular functions. Here we will build on these results to investigate the mechanism of allostery in Gsp1 and assess its generality in other GTPases, with implications for understanding mechanisms of disease mutations and for development of modulators. We also plan to test our model of GTPase regulation by determining quantitative cellular consequences of fine-tuned perturbations to GTPases regulators. Future directions include expansion of these perturbation measurements to other central biological switches. The uncovered principles of cellular control can guide cellular engineering, and in conjunction with computationally designed new functions may ultimately lead to new ways to counteract misregulation in disease.