STRUC & MECHANISTIC ANALYSIS OF SIGNALING MOL INVOLVED IN BIOFILM FORMATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 细菌第二信使双-（3 "-5”）-环二聚鸟苷一磷酸（c-di-GMP）调节复杂的生物过程，如细胞运动性，生物膜形成和毒力。催化c-di-GMP、GGDEF和EAL结构域的合成和降解的蛋白质结构域分别在迄今为止测序的几乎每种微生物基因组中被大量鉴定。我们对这些酶的功能和调节的理解仅处于起步阶段。利用结构生物学结合酶的测定，我们希望破译的监管机制和关键酶控制生物膜形成和细胞毒性的操作模式，使用条件致病菌铜绿假单胞菌作为模型系统。其结果将具有广泛的应用，例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型生物医学疗法。在更一般的条件下，我们感兴趣的是在多结构域蛋白质的分子内和分子间的调节原则，包括变构和反馈控制，在信号转导和酶的调节的基本问题。 我们获得了三种蛋白质的晶体含有EAL-，GGDEF-或串联结构域在各种条件下。在CHESS，光束线F1进行的初步X射线衍射实验显示出有希望的结果。晶体衍射X射线的最大分辨率为10埃，但我们相信，改进的结晶和冷冻条件将提高衍射极限。由于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.