PULSED DIPOLAR SPECTROSCOPY STUDY ON THE DIGUANYLATE CYCLATE WSPR

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， NCRR赠款不直接向子项目或子项目工作人员提供资金。 触发细菌发病机制和生物膜形成的环境信号是由环二聚鸟苷一磷酸（c-di-GMP）（一种独特的真细菌第二信使）水平的变化介导的。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，它们分别负责其产生和降解。二鸟苷酸环化酶WspR是革兰氏阴性菌中保守的含有GGDEF结构域的反应调节剂，并且其在抑制位点处结合至c-di-GMP。WspR的过表达导致超生物膜形成，而功能丧失突变体显示生物膜形成和细胞毒性降低。 来自铜绿假单胞菌的全长二鸟苷酸环化酶WspR在其抑制状态下的晶体结构最近在康奈尔大学的Holger Sondermann教授的小组中解析。然而，X射线晶体学在研究溶液中出现的WspR的低聚状态时遇到困难，因为在这种实验中使用的高浓度下，蛋白质自发地形成四聚体。 我们用脉冲偶极ESR谱（PDS）研究了二鸟苷酸环化物WspR不同寡聚（功能）态的结构。顺磁自旋标记（MTSSL）连接到一个单一的蛋白质分子的不同结构域中的天然积累的半胱氨酸残基。单个WspR分子在CheY结构域的49位和GGDEF结构域的240位含有两个半胱氨酸残基。为了减少这种蛋白质的二聚体和四聚体状态中的自旋标记的数量，从而避免结果解释的复杂性，进行了两种替代性点突变：C49 S或C240 S，并测量附着于剩余半胱氨酸的自旋标记之间的距离。

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.