Investigation of bacterial adhesion pili via novel Brownian dynamics simulations

描述（由申请人提供）：分子内运动在多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，这些运动已经证明很难研究实验和计算。标准的计算技术仅限于纳秒范围，而上述运动发生在微秒到毫秒之间。或者，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，从计算的角度来研究分子内运动，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BD模拟中的分子内运动。我们的方法包括：识别蛋白质结构域，将它们作为BD框架中的独立刚体处理，并通过经验能量函数将它们束缚。该方法，系留布朗动力学（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的灵活性。TBD最初将根据计算数据进行测试，例如正常模式分析，然后通过利用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I类粘附皮利在肾盂肾炎E。杆菌这些结构对于粘连是必要的和足够的，这是感染的重要步骤。我们将开发的技术是必要的调查皮利的形成，因为它已被假设后，通过引导蛋白在细菌外膜的内部铰链弯曲运动的单体单元扩散需要有效地对接在其正确的构象的蛋白质。在验证了我们的TBD算法后，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。我们的模拟将描述一个原子尺度的菌毛组装模型，可以使识别的药理学靶点的菌毛形成和感染的抑制。公共卫生相关性在这项研究中，将开发允许研究柔性蛋白质的计算模型，并用于研究I类皮利的形成，I类皮利是细菌产生的能够粘附到人类宿主细胞的结构。通过阐明这些结构形成的分子机制，我们希望告知破坏皮利组装的策略，从而防止或禁用粘附和感染。

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.