PULSED DIPOLAR SPECTROSCOPY STUDY ON THE DIGUANYLATE CYCLATE WSPR

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 触发细菌致病和生物被膜形成的环境信号是由环二聚鸟苷(c-di-GMP)水平的变化介导的，环二聚鸟苷是一种独特的真细菌第二信使。细胞c-di-GMP结构域浓度的严格调节由二鸟苷酸环化酶和磷酸二酯酶控制，这两种酶分别负责其产生和降解。地鸟苷环化酶WSPR是革兰氏阴性菌中一种保守的含GGDEF结构域的反应调节因子，它与c-di-GMP结合在一个抑制部位。WSPR的过表达会导致超生物膜的形成，而功能丧失的突变体则表现出生物膜形成和细胞毒性的减少。 最近，康奈尔大学的Holger Sondermann教授团队对铜绿假单胞菌全长二鸟苷环化酶WSPR的晶体结构进行了研究。然而，X射线结晶学很难研究WSPR在溶液中出现的低聚状态，因为在这样的实验中，在高浓度下，蛋白质自发形成四聚体。 我们用脉冲偶极ESR波谱(PDS)研究了二鸟苷环化合物WSPR的不同低聚(官能态)结构。顺磁自旋标记(MTSSL)被附着在单个蛋白质分子不同区域中自然积累的半胱氨酸残基上。一个WSPR分子含有两个半胱氨酸残基，分别位于Chey结构域的49位和GGDEF结构域的240位。为了减少该蛋白质二聚体和四聚体状态下的自旋标记物的数量，从而避免解释结果的复杂性，进行了两个可选的点突变：C49S或C240S，并测量了附着在剩余半胱氨酸上的自旋标记物之间的距离。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP domain concentration is governed by diguanylate cyclases and phosphadiesterases, which are responsible for its production and degradation, respectively. Diguanylate cyclase WspR is a conserved GGDEF domain-containing response regulator in Gram-negative bacteria and it is bound to c-di-GMP at an inhibitory site. Overexpression of WspR causes hyperbiofilm formation, whereas loss-of-function mutants show reduced biofilm formation and cytotoxicity. The crystal structure of full length Diguanylate cyclase WspR from P. aeruginosa in its inhibitory state was recently resolved in the group of Professor Holger Sondermann, Cornell University. However, X-ray crystallography experiences difficulties to study the oligomeric states of WspR that appear in solution since at high concentration, used in such an experiment, the protein spontaneously forms tetramer. We used pulsed dipolar ESR spectroscopy (PDS) to elucidate the structure of differenr oligomeric (functional) states of Diguanylate Cyclate WspR. Paramagnetic spin-labels (MTSSL) were attached to naturally accruing cysteine residues in different domains of a single protein molecule. A single WspR molecule contains two cysteine residues at positions 49 in CheY domain and 240 in GGDEF domain. In order to reduce the number of spin-labels in the dimeric and tetrameric state of this protein and consequently to avoid a complication in the interpretation of results, two alternative point mutations were done: C49S or C240S, and distances between spin-labels attached to the remaining cysteines were measured.