STRUC & MECHANISTIC ANALYSIS OF SIGNALING MOL INVOLVED IN BIOFILM FORMATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细菌第二信使双(3‘-5’)-环二聚鸟苷一磷酸(c-di-GMP)调节细胞运动、生物膜形成和毒力等复杂的生物过程。到目前为止，几乎所有的微生物基因组序列中都发现了催化合成和降解c-di-GMP、GGDEF和EAL结构域的蛋白质结构域。我们对这些酶的功能和调节的了解还处于初级阶段。我们希望以条件致病菌铜绿假单胞菌为模型系统，利用结构生物学和酶分析相结合的方法，破译控制生物被膜形成和细胞毒性的关键酶的调控机制和作用模式。这些结果将有广泛的应用，如开发用于治疗传染病的新型抗生素，优化生物技术过程，或开发新的生物医学疗法。更广泛地说，我们感兴趣的是多结构域蛋白质的分子内和分子间调节原理，包括变构和反馈控制，信号转导和酶调节的基本问题。 我们在不同的条件下获得了三种含有EAL-、GGDEF-或串联结构域的蛋白质的晶体。在CHESS，BEAM LINE F1上进行的初步X射线衍射实验显示了令人振奋的结果。晶体使X射线的最大分辨率达到10埃，但我们相信，改进的结晶和冷冻条件将提高衍射极限。考虑到一些晶体的尺寸很小，我们需要一个明亮的光源来进行晶体筛选和数据收集。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The bacterial second messenger bis-(3¿¿"-5¿¿")-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates complex biological processes such as cell motility, biofilm formation and virulence. Protein domains catalyzing synthesis and degradation of c-di-GMP, GGDEF and EAL domains, respectively, have been identified in large numbers in almost every microbial genome sequenced to date. Our understanding regarding function and regulation of these enzymes is only in its infancy. Using structural biology in combination with enzymatic assays, we hope to decipher the regulatory mechanisms and modes of operation of key enzymes controlling biofilm formation and cytotoxicity, using the opportunistic pathogen Pseudomonas aeruginosa as a model system. Results will have widespread applications such as the development of novel antibiotics for the treatment of infectious diseases, optimization of biotechnological processes, or the development of novel biomedical therapies. In more general terms, we are interested in the intra- and intermolecular regulatory principles in multi-domain proteins, including allosteric and feedback control, fundamental questions in signal transduction and enzyme regulation. We obtained crystals for three proteins containing EAL-, GGDEF- or tandem-domains in various conditions. Preliminary X-ray diffraction experiments performed at CHESS, beamline F1 show promising results. Crystals diffract X-rays to a maximum resolution of 10 angstrom but we are confident that improved crystallization and freezing conditions will increase the diffraction limit. Given the small size of some of the crystals, we require a bright light source for crystal screening and data collection.