TrHbs: Biophysical Consequences of a Nonpolar Tunnel

描述(申请人提供)：截断血红蛋白(TrHbs)是最近发现的一类小的氧结合原血红蛋白，广泛分布于原核生物中。许多出现在对人致病的细菌中(例如结核分枝杆菌、禽类分枝杆菌、麻风分枝杆菌、白喉科尼杆菌、百日咳杆菌、嗜肺军团菌、金黄色葡萄球菌和炭疽杆菌)。该项目将侧重于结核病的两种trHb--trHbN和trHbO。TrHbN保护有氧呼吸免受一氧化氮的抑制作用，并增强传染性。TrHbO的功能是细菌生长所必需的。由于trHbs不存在于人类体内，这使它们成为潜在的药物靶点。这个项目的重点是探索这两个trHb的不寻常的生物物理性质，这些性质被认为是具有潜在生物医学意义的不寻常功能的基础。设计了几个特定的目标，以揭示在远端血袋中发现的氢键网络的性质和意义，以及将溶剂与血红素的配体结合位置连接起来的大型非极隧道的性质和意义。为了深入了解这两个重要蛋白质的氢键网络和非极通道的功能，将使用几种技术，包括光解、停流光谱、振动光谱(FTIR、UV和可见光共振拉曼)、结构分析(X-射线衍射、核磁共振)、定点突变和构象捕获以及配体重新结合的动力学。与整个项目的这一部分并行，将使用遗传和分子生物学方法将生物物理结果与体内功能研究相关联。

DESCRIPTION (provided by applicant): Truncated hemoglobins (trHbs) are a recently discovered class of small oxygen-binding protohemeproteins, widely distributed in prokaryotes. Many occur in bacteria pathogenic to man (e.g. Mycobacterium tuberculosis (TB), Mycobacterium avium, Mycobacterium leprae, Cornybacterium diphtheriae, Bordetella pertussis, Legionella pneumophila, Staphylococcus aureus and Bacillus anthracis). This project will focus on trHbN and trHbO, the two trHbs from TB. TrHbN protects aerobic respiration from the inhibitory action of nitric oxide and enhances infectivity. The function oftrHbO is essential for bacterial growth. Since trHbs are not present in man, this makes them potential drug targets. The emphasis of this project is on probing those unusual biophysical properties of these two trHbs that are hypothesized to be the basis for the unusual functionalities that are of potential biomedical significance. Several specific aims are designed to expose the nature and significance of the hydrogen bonding network found in the distal hempocket and of the large apolar tunnel that links the solvent to the ligand binding site at the heme. To achieve in depth insight into how the hydrogen bonding network and the apolar tunnel functioning of these two important proteins, several techniques will be used including photolysis, stopped-flow spectrometry, vibrational spectroscopy (FTIR, UV and visible resonance Raman), structure analysis (X-ray diffraction, NMR), site directed mutagenesis and conformational trapping and kinetics of ligand rebinding. In parallel with this part of the overall project, genetic and molecular biological approaches will be used to correlate the biophysical results with the in vivo functional studies.