BRIGE: Hierarchical Design of Membrane-Based Mechanotransduction Systems

细胞表面存在着一个非常复杂的蛋白质、激素和其他溶质的混合物，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定的跨膜和细胞内蛋白质的相互作用，从而导致膜曲率和细胞形状的改变。其中一个例子是BAR结构域，它是一个二聚化、膜结合和曲率传感模块家族，通过响应细胞外配体结合调节膜结构，在机械转导中发挥不同的作用。BAR结构域的一个独特特征是它们能够在二聚化时以高亲和力结合磷脂，从而赋予高度的正或负膜曲率。因此，在生物信号输入（配体结合）和生物力学信号输出（膜曲率）之间存在着复杂的联系，我们将利用这一联系设计一种基于膜的生物传感器，以检测复杂生物混合物中的小分子毒素双酚a （BPA）。在我们的设计中，配体诱导的FBAR结构域二聚化将驱动膜结构的变化，这种变化可以通过双折射和膜电导来量化。目标1 -生物信号输入（配体结合）：利用综合计算和遗传选择方法，我们将重新设计人类雌激素受体<e:1> (her<e:1>)配体结合域（LBD），以高亲和力和选择性地结合BPA。我们最初的对接研究表明BPA能够结合her<e:1> LBD，这将指导我们使用基于酵母的her<e:1>激活转录试验来修饰的初始残基选择。目标2 -生物信号输出（膜结合）：利用结构导向设计，我们将开发一系列“分子尺子”，将我们设计的her<s:1> lbd连接到FBAR结构域。螺旋连接体将使配体结合时发生的构象变化传播到FBAR单体，从而驱动二聚化和随后的膜结合。我们将使用一系列膜结合（双折射）和寡聚化（PFO-PAGE）来评估连接体结构和长度对机械转导的影响。目标3 -膜结构的量化：我们将采用一系列互补的光谱技术来确定我们设计的机械转导受体的相变。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）将用于成像所得的膜结构。智力优势：我们设计基于膜的生物传感器的集成方法的一个主要优势是低成本，直接制造生物相容性，无标签系统，可以适应各种毒素。此外，我们的设计结果将为膜介导的机械转导的结构基础提供详细的分子见解。使用脂质立方相（LCP）作为膜环境，可以通过膜电导或双折射的变化在连续流动条件下进行无标记检测。lcp可以通过简单的混合和孵育制备，对广泛的温度和溶质都很稳定，并提供可用于原位生物传感器应用的生物相容性基质。更广泛的影响：BPA是一种重要的环境毒素，与许多神经系统疾病和癌症的风险增加有关，然而，环境中暴露水平与其生物效应之间的定量联系仍然是一个活跃的研究领域。我们提出的设计将使人们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，我们的工作还将有助于加深对纳米技术在生物技术中的应用的理解，并通过在利哈伊大学的实习，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.