BRIGE: Hierarchical Design of Membrane-Based Mechanotransduction Systems

主要研究者：Berger，Bryan W.Proposal Number：1227924细胞表面存在于蛋白质、激素和其他溶质的非常复杂的混合物中，这些溶质通过结合特定的细胞表面受体来传递信号。在机械转导中，与细胞表面受体结合的配体驱动与特异性跨膜和细胞内蛋白的相互作用，这导致膜曲率和细胞形状的变化。一个这样的实例是BAR结构域，其是二聚化、膜结合和曲率传感模块的家族，其通过响应于细胞外配体结合调节膜结构而在机械转导中发挥不同的作用。BAR结构域的独特特征是它们在二聚化时以高亲和力结合磷脂的能力，从而赋予高度的正或负膜曲率。因此，生物信号输入（配体结合）和生物力学信号输出（膜曲率）之间存在着错综复杂的联系，我们将使用这种联系来设计基于膜的生物传感器，以从复杂的生物混合物中检测小分子毒素双酚A（BPA）。在我们的设计中，配体诱导的FBAR结构域的二聚化将驱动膜结构的变化，其可以通过双折射以及膜电导来量化。我们的方法可以按照以下具体目标来描述：目标1 -生物信号输入（配体结合）：使用集成的计算和遗传选择方法，我们将重新设计人雌激素受体α（hER α）配体结合结构域（LBD），以高亲和力和选择性结合BPA。我们最初的对接研究表明BPA能够结合hER á LBD，这将指导我们使用基于酵母的转录测定对hER á活化进行修饰的残基的初步选择。目标2 -生物信号输出（膜结合）：使用结构导向设计，我们将开发一系列“分子统治者”来将我们设计的hER á LBD连接到FBAR结构域。 螺旋接头将使配体结合时发生的构象变化能够传播到FBAR单体，驱动二聚化和随后的膜结合。我们将使用一系列的膜结合（双折射）和寡聚化（PFO-PAGE）来评估接头结构和长度对机械transduction.Aim 3 -膜结构的定量：我们将使用一系列互补的光谱技术来确定我们设计的机械transductionreceptor的相变。线性红外二色性（LID）和循环伏安法将用于测量受体诱导的膜相变的结构和动力学，电子显微镜（EM）将用于对所得膜结构进行成像。我们设计基于膜的生物传感器的集成方法的主要优点是低成本、简单地制造生物相容的，无标记系统，可适用于各种毒素。此外，我们的设计结果将提供详细的，分子洞察膜介导的机械转导的结构基础。作为膜环境的无标记立方相（LCP）的使用允许在连续流动条件下通过膜电导或双折射的变化进行无标记检测。LCP可以通过简单的混合和孵育来制备，对广泛的温度和溶质具有鲁棒性，并提供可用于原位生物传感器应用的生物相容性基质。BPA是一种重要的环境毒素，与多种神经系统疾病和癌症的风险增加有关，然而，环境中的接触水平与其生物效应之间的定量联系仍然是一个积极研究的领域。我们提出的设计将能够详细了解这种联系，并有助于改进监测环境中毒素的方法。通过与北安普顿县社区学院合作开发教学模块，我们的工作也将有助于更好地了解纳米技术在生物技术中的应用，并通过在利哈伊大学实习扩大学生对工程研究和教育的参与。

PI: Berger, Bryan W.Proposal Number: 1227924The cell surface exists in a remarkably complex mixture of proteins, hormones and other solutes that communicate signals through binding specific cell surface receptors. In mechanotransduction, ligand binding to a cell surface receptor drives interactions with specific transmembrane and intracellular proteins, which results in a change in membrane curvature and cell shape. One such example are BAR domains, which are a family of dimerization, membrane binding, and curvature sensing modules that play diverse roles in mechanotransduction through modulating membrane structure in response to extracellular ligand binding. A unique feature of BAR domains is their ability to bind phospholipids with high affinity upon dimerization, thereby imparting a high degree of positive or negative membrane curvature. Thus, there is an intricate link between the biological signal input (ligand binding) and biomechanical signal output (membrane curvature) that we will use to design a membrane-based biosensor to detect the small molecule toxin bisphenol-A (BPA) from complex, biological mixtures. In our design, ligand induced dimerization of FBAR domains will drive changes in membrane structure that can be quantified by birefringence as well membrane conductance. Our approach can be described in terms of the following Specific Aims:Aim 1 - Biological Signal Input (Ligand Binding): Using an integrated computational and genetic selection approach, we will redesign the human estrogen receptor á (hERá) ligand binding domain (LBD) to bind BPA with high affinity and selectivity. Our initial docking studies indicate BPA is capable of binding hERá LBD, which will guide our initial choice of residues to modify using a yeast-based transcriptional assay for hERá activation.Aim 2 - Biological Signal Output (Membrane Binding): Using structure-guided design, we will develop a series of "molecular rulers" to link our designed hERá LBDs to FBAR domains. The helical linkers will enable conformational changes that occur upon ligand binding to propagate to FBAR monomers, driving dimerization and subsequent membrane binding. We will use a series of membrane binding (birefringence) and oligomerization (PFO-PAGE) to assess the effects of linker structure and length on mechanotransduction.Aim 3 - Quantification of Membrane Structure: We will a series of complementary spectroscopic techniques to determine the phase transitions that underlie our designed mechanotransduction receptor. Linear infrared dichroism (LID) and cyclic voltammetry will be used to measure the structure and kinetics of receptor-induced membrane phase transitions, and electron microscopy (EM) will be used to image the resultant membrane structures.Intellectual Merit: A major advantage of our integrated approach to designing a membrane based biosensor is the low-cost, straightforward fabrication of a biocompatible, label-free system that can be adapted to a wide range of toxins. Furthermore, the results of our design will provide detailed, molecular insight into the structural basis that underlies membrane-mediated mechanotransduction. The use of a lipidic cubic phase (LCP) as the membrane environment allows label-free detection under continuous flow conditions through changes in membrane conductance or birefringence. LCPs can be prepared through simple mixing and incubation, are robust to a wide range of temperatures and solutes and provide a biocompatible matrix that can be used for in situ biosensor applications.Broader Impact: BPA is an important environmental toxin linked to increased risk for numerous neurological disorders as well as cancers, yet a quantitative link between exposure levels in the environment and their biological effects remains an area of active research. Our proposed design will enable a detailed understanding of this linkage and contribute to improved methods for monitoring toxins in the environment. Through developing teaching modules in partnership with Northampton County Community College, out work will also contribute to a greater understanding of nanotechnology applications in biotechnology, and broaden participation of students in engineering research and education through internships at Lehigh University.