BRIGE: Hierarchical Design of Membrane-Based Mechanotransduction Systems

Lehigh University, Bethlehem PA

Investigators

Abstract

PI: Berger, Bryan W. Proposal Number: 1227924 The 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.