Monitoring and Tuning a Gas-Binding Heme Protein with Unnatural Amino Acids

项目总结。 血红素一氧化氮和/或氧结合蛋白(H-NOX)形成一类气体敏感蛋白，参与 结合气体和启动信号级联在原核和真核细胞与关键成员 这个类是可溶性鸟苷环化酶(SGC)的NO结合的血红素结构域，是人体内主要的NO感受器。 人类。非天然氨基酸(UAA)是化学合成的分子，只需稍作改动即可 天然氨基酸已被用来帮助研究各种生物和医学上的相关 蛋白质系统。UAA具有独特的优势，即可针对以下应用进行化学定制 兴趣和位点特异性地结合到蛋白质中，同时对蛋白质结构的侵袭最小，因为 尿酸本身并不比天然氨基酸大多少。这个项目使用了琥珀密码子 在H-NOX蛋白中加入非天然氨基酸研究构象变化的方法学 并调整气体结合亲和力。这个项目的第一个目标是利用含有丁腈的振动报告UaAs 或叠氮基团监测两个细菌H-NOX的溶剂化环境和构象变化 蛋白质。该项目的第二个目标是调节O2-结合的氧结合亲和力 利用UAAS产热厌氧菌产H-NOX。这项研究使用了许多生物物理学 技术(FTIR、停流紫外可见光谱、激光闪光光解、CD光谱和X射线 结晶学)分析掺入UAA的H-NOX突变体。这些研究将延长我们的 了解这些重要的气体结合蛋白，致力于开发一种新的氧气输送分子 在治疗缺氧相关疾病方面具有潜在的用途，并扩大了其应用和 非天然氨基酸在研究和/或设计其他生物医学结构和功能方面的应用 相关蛋白质。

PROJECT SUMMARY. The heme nitric oxide and/or oxygen binding (H-NOX) proteins form a class of gas sensing proteins involved in binding gases and initiating signaling cascades in both prokaryotic and eukaryotic cells with a key member of this class being the NO-binding heme domain of soluble guanylate cyclase (sGC), the main NO sensor in humans. Unnatural amino acids (UAAs) are chemically synthesized molecules with minor alterations from naturally occurring amino acids that have been used to help study various biologically and medically relevant protein systems. UAAs have the distinct advantage of being chemically customizable for an application of interest and site-specifically incorporated into proteins while being minimally invasive of protein structure as the UAAs themselves are not much larger than native amino acids. This project utilizes the amber codon methodology to incorporate unnatural amino acids in H-NOX proteins to both study conformational changes and tune gas-binding affinity. The first aim of this project is to utilize vibrational reporter UAAs containing nitrile or azido groups to monitor solvation environments and conformational changes of two bacterial H-NOX proteins. The second aim of this project is to tune the oxygen-binding affinity of the O2-binding Thermoanaerobacter tencongensis H-NOX using UAAs. This research employs a number of biophysical techniques (FTIR, stopped-flow UV-Visible spectroscopy, laser flash photolysis, CD spectroscopy, and X-ray crystallography) to analyze the UAA-incorporated H-NOX mutants. These studies will extend our understanding of these important gas-binding proteins, work towards developing a new O2 delivery molecule with potential use as a therapeutic agent to treat hypoxia-related diseases, and expand the applications and utility of unnatural amino acids to study and/or engineer the structure and function of other biomedically relevant proteins.