Investigation of bacterial adhesion pili via novel Brownian dynamics simulations

描述（由申请人提供）：分子内运动在各种生物过程中发挥重要作用，包括信号转导，催化和分子马达。不幸的是，这些运动已经被证明很难在实验和计算上进行研究。标准的计算技术仅限于纳秒级，而上述运动发生在微到毫秒级。另外，布朗动力学（BD）算法可以达到必要的时间尺度，但传统上仅限于刚性结构。因此，为了从计算的角度研究分子内运动，必须开发新的技术。在这个建议中，我们将开发和实施计算策略，包括分子内运动在BD模拟。我们的方法包括：识别蛋白质结构域，将它们视为BD框架中的独立刚体，并通过经验能量函数将它们捆绑在一起。该方法，系缚布朗动力学（TBD），将允许长时间尺度的模拟，包括多结构域蛋白质的分子内灵活性。TBD将首先针对正常模式分析等计算数据进行测试，然后利用Taq DNA聚合酶的中子散射光谱进行改进。然后，我们将应用这些技术来研究肾盂肾炎大肠杆菌中I类粘连菌毛的形成。这些结构对于粘连是必要和充分的，这是感染的重要一步。我们将开发的技术对于研究菌毛的形成是必要的，因为它已经被假设，当单体单元通过细菌外膜中的引导蛋白扩散后，需要内部的铰链弯曲运动来有效地将蛋白质固定在其正确的构象中。在验证了我们的TBD算法之后，我们将测试这个对接假设，并试图确定几个突变的结构效应的分子机制。我们的模拟将描述毛菌组装的原子尺度模型，这可能使识别抑制毛菌形成和感染的药理学靶标成为可能。在这项研究中，允许研究柔性蛋白的计算模型将被开发并用于研究I类菌毛的形成，细菌产生的结构使其能够粘附于人类宿主细胞。通过阐明这些结构形成的分子机制，我们希望为破坏毛组装的策略提供信息，从而预防或阻止粘连和感染。

DESCRIPTION (provided by applicant): Intramolecular motions play an important role in a variety of biological processes including signal transduction, catalysis, and molecular motors. Unfortunately, these motions have proven difficult to study both experimentally and computationally. Standard computational techniques are limited to the nanosecond regime while the above motions occur in micro- to milliseconds. Alternatively, Brownian dynamics (BD) algorithms can reach the necessary timescales, but have traditionally been limited to rigid structures. Therefore, to investigate intramolecular motions from a computational perspective, novel techniques must be developed. In this proposal, we will develop and implement computational strategies to include intramolecular motions in BD simulations. Our approach involves: identifying protein domains, treating them as separate rigid bodies in a BD framework, and tethering them via an empirical energy function. The method, Tethered Brownian dynamics (TBD), will allow long timescale simulations that include intramolecular flexibility of multi-domain proteins. TBD will be initially tested against computational data such as normal mode analysis but then refined by utilizing neutron scattering spectra of Taq DNA polymerase. We will then apply these techniques to study the formation of class I adhesion pili in pyelonephritic E. coli. These structures are necessary and sufficient for adhesion, an important step towards infection. The techniques we will develop are necessary for investigating pili formation because it has been hypothesized that after the monomeric units diffuse through an usher protein in the bacterial outer membrane an internal hinge-bending motion is required to effectively dock the protein in its proper conformation. After validating our TBD algorithm for this system, we will test this docking hypothesis and attempt to identify the molecular mechanism for the structural effects of several mutations. Our simulations will describe an atomic scale model for pilus assembly that may enable the identification of pharmacologic targets for the inhibition of pilus formation and infection. PUBLIC HEALTH RELEVANCE In this study, computational models that allow the study of flexible proteins will be developed and used to investigate the formation of class I pili, structures that bacteria produce to enable adhesion to human host cells. By elucidating the molecular mechanism of the formation of these structures, we hope to inform strategies to disrupt pili assembly, thus preventing or disabling adhesion and infection.