Computational design of proteins and protein functions

项目概要/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， 分子/细胞工程策略，以揭示生物调节的原则。该提案结合了 我们在这些领域的两个NIGMS赠款。 在我们的计算蛋白质设计的工作中，我们已经计算工程蛋白质， 响应细胞中新的小分子信号，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细胞过程。我们也有先进的方法， 完全从头开始设计具有精确可调形状的蛋白质。这项拟议中的工作是以我们的新方法为基础的 为了解决一个核心的未解决的挑战，同时设计从头蛋白质和用户的几何形状， 定义的功能性位点被放置在其中，具有针对功能优化的原子精度。这项工作应该大大 拓展了新功能的可设计空间。我们计划将重新设计的蛋白质整合到 模块化系统可以控制生物行为以响应新的信号。 我们在天然蛋白质功能方面的工作旨在了解中央调节蛋白如何在 互联的细胞网络，以及这些网络如何在突变等扰动下发生变化。 我们研究了一个由相反调节器控制的双态开关（一个GTdR），因为这个基序在 生物学通过系统诱变GTsp 1和整合系统测量， 规模（遗传相互作用映射）与生物物理学，我们发现了以前未知的变构网站上的 GTT是其功能的核心。此外，我们的研究结果提出了一个新的模型，即多效性GTsp 1 差异调节不同的细胞功能。在这里，我们将建立在这些结果，以调查机制 Gsp 1的变构，并评估其在其他GTP酶的普遍性，与理解机制的影响， 疾病突变和调节剂的开发。我们还计划测试我们的GTTT监管模型 通过确定对GTP酶调节剂的微调扰动的定量细胞后果。未来 方向包括将这些扰动测量扩展到其它中心生物开关。的 细胞控制的未被揭示的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导致新的方法来对抗疾病中的失调。

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.