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

项目摘要。 血红素一氧化氮和/或氧结合（H-NOX）蛋白形成一类涉及以下的气敏蛋白： 在原核和真核细胞中结合气体和启动信号级联， 这一类是可溶性鸟苷酸环化酶（sGC）的NO结合血红素结构域，sGC是脑内主要的NO感受器， 人类非天然氨基酸（UAA）是化学合成的分子， 天然存在的氨基酸已被用于帮助研究各种生物学和医学相关的 蛋白质系统UAA具有可化学定制以用于以下应用的明显优势： 感兴趣和位点特异性地掺入蛋白质，同时对蛋白质结构的侵入性最小， UAA本身并不比天然氨基酸大多少。这个项目利用琥珀密码子 在H-NOX蛋白中掺入非天然氨基酸以研究构象变化的方法 并调节气体结合亲和力。本项目的第一个目标是利用含腈的振动报告子UAA 或叠氮基团来监测两种细菌H-NOX的溶剂化环境和构象变化 proteins.该项目的第二个目的是调节O2结合的氧结合亲和力， 腾冲嗜热厌氧菌H-NOX使用UAA。这项研究采用了一些生物物理 技术（FTIR，停流紫外可见光谱，激光闪光光解，CD光谱，和X射线 晶体学）以分析UAA掺入的H-NOX突变体。这些研究将扩大我们的 了解这些重要的气体结合蛋白，致力于开发一种新的O2输送分子 具有作为治疗剂治疗缺氧相关疾病的潜在用途， 利用非天然氨基酸来研究和/或工程化其他生物医学上的结构和功能， 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.