Dynamic behavior of a transmembrane molecular switch as an artificial cell-surface receptor

Abstract

Supramolecular functions of a novel synthetic amphiphile with an azobenzene core interposed between two aminoalkyl chains as an artificial transmembrane receptor were investigated. Hybrid vesicular membrane was prepared in combination with the receptor and a bilayer-forming lipid dimyristoylphosphatidylcholine (DMPC). Aggregation behavior of the receptor embedded in the bilayer membrane was evaluated spectrophotometrically by monitoring stacking of the azobenzene moiety. Upon addition of 1-hydroxy-2-naphthaldehyde as an external signal ligand, it was effectively recognized at both ends of the receptor by forming the corresponding Schiff's base, and followed by phase reorganization of the receptor in the membrane. The resulting receptor–ligand complex provided an appropriate binding site for copper(II) ions, which may act as a second messenger signal. Signal transduction behavior of the present supramolecular system was examined by employing NADH-dependent lactate dehydrogenase (LDH) as an effector. We found that the transmembrane receptor is capable of performing as a molecular switch for the enzymatic reaction.

Introduction

Signal transduction, or cell communication is simply the mean by which natural cells respond to signals coming from outsides. The purpose of the signal transduction is functional coordination within the cell, between cells or between organs, allowing them to respond to external environment. Many studies have been carried out to understand mechanisms that mediate inter- or intra-cellular signal transduction. Up to the present time, it has been clear that signal transduction is generally accomplished by way of one or more of the following processes: receptor–ligand interaction, ion channeling, second messenger pathway, and transcription of a desired gene product [1]. However, receptor functions remain to be clarified at the molecular level with emphasis on how external signals are recognized, received and transported through the cell membrane to target molecules inside the cells. Thus, the approach to illustrate the principle of signal transduction based on molecular recognition by artificial receptors is one of the most attractive subjects in supramolecular chemistry [2].

So far, we have attempted to simulate and simplify the signal transduction system [3], [4], [5], [6], which involves ligand–receptor interaction and G-protein-linked pathway. In this process, binding of an external signal to a receptor embedded in the cell membrane results in activation of the G-protein as a second messenger, which conveys the message to the next effector such as an enzyme in the signaling pathway. Various types of artificial receptors capable of recognizing organic signaling ligands in bilayer membranes have been developed in our research group. For example, a bile acid derivative having an amino group as an artificial cell-surface receptor transmits an external signal to an enzyme in collaboration with a transmitter [6]. The bile acid derivative acts as a host capable of recognizing both aromatic aldehydes and copper(II) ions. On the other hand, lactate dehydrogenase (LDH) binds to the bilayer membrane surface [7], [8] and the LDH activity is specifically inhibited by copper(II) ions. Thus, we have successfully simulated signal transduction on the lipid membrane by harmonic constitution of the following components: an artificial receptor, an aromatic aldehyde, a bilayer-forming lipid, copper(II) ions and LDH.

In living cells, however, most signal transduction involves more complex events. For example, cell-surface receptor proteins act as signal transducers. They bind the signaling ligand and convert this extracellular event into one or more intracellular signals that alter the cell function. A kind of G-protein linked receptor responds to light as photoreceptor; rhodopsin receives a photon of light and transduces that to an electrical signal [1]. In order to construct a more organized supramolecular system focused on the transmission of an extra-vesicular signal to the inside of a membrane vesicle and the photo-sensitivity, we have designed and synthesized a novel ampihiphile Azo(C₁₂N)₂ as an artificial transmembrane receptor (Fig. 1). This molecule is composed of two amino groups as an external signal binding site and an intracellular regulating site, an azobenzene core as a photosensitive part, and alkyl chains as transmembrane parts. Aggregation behavior and microenvironment of the receptor in membrane can be evaluated spectrophotometrically by monitoring stacking of the azobenzene moiety [9]. In addition, the aggregation of the azobenzene moiety, which affects the affinity towards the signal molecules, can be controlled by irradiation of light, since azobenzene derivatives generally cause reversible conformational changes through photo-induced cis/trans isomerization. The reversibility of signaling in cells is another important characteristic for signal transduction. Thus, the isomerization of azobenzene moiety in the artificial receptor may enable to control the signal transduction flow reversibly. In this paper, we report basic behavior of signal transduction ability of the system proposed in Fig. 1. Such an artificial signal transduction system may find novel application in numerous fields, such as medicine, diagnostics, nanotechnology, and artificial intelligence.