Binding assays with artificial tethered membranes using surface plasmon resonance

doi:10.1016/j.ymeth.2006.05.007

Methods

Volume 39, Issue 2, June 2006, Pages 134-146

https://doi.org/10.1016/j.ymeth.2006.05.007 Get rights and content

Abstract

Surface sensitive optical techniques based on surface plasmon resonance have become interesting for biosciences in the context of biorecognition and binding studies at functional surfaces. We use surface plasmon resonance spectroscopy (SPS) in combination with surface plasmon enhanced fluorescence spectroscopy (SPFS) for the characterization of interaction processes associated with biomembranes. The biological membrane is mimicked by a tethered membrane consisting of a planar lipid bilayer attached to a gold surface via a hydrophilic anchor peptide. The interaction between membrane-bound hydrophobic compounds and free hydrophilic molecules is monitored in real-time and with high sensitivity and selectivity by combined SPS/SPFS. In this review we shortly discuss the principles of surface plasmon resonance and its utilization in SPS and SPFS. A detailed description of the required instrumentation for combined SPS and SPFS is presented. Furthermore, we outline the design of a binding assay with a tethered bilayer and the procedure of the artificial membrane system built-up is delineated. We also present examples that demonstrate the potential of combined SPS/SPFS assays with artificial tethered membranes. The method provides insight into the interaction of integral membrane proteins with various hydrophilic ligands and the specific recognition of small lipophilic molecules by soluble proteins.

Introduction

Biomembranes are vital for complex biological processes such as selective transport of components into and out of the cell, cell signaling, physical and functional connection to other cells, infections by pathogenic organisms or cell adhesion processes. Many of these processes rely on the interaction of membrane-bound components, mostly membrane proteins, with their (soluble) interaction partners.

The lipid bilayer of cellular membranes provides the natural environment for integral membrane proteins and membrane lipids. Hence, detailed investigations of membrane-related processes require the development of suitable in vitro systems mimicking biomembranes to preserve their native structural and functional properties. On the other hand, the complexity of a biomembrane has to be reduced if a specific receptor/ligand interaction is to be analyzed. Tethered artificial membranes represent a strategy to simplify complicated membrane processes to single interactions between selected proteins, peptides and lipids. Tethered membranes are model membranes composed of a solid substrate, a tethering layer and a lipid bilayer [1], [2]. They facilitate the incorporation of membrane proteins or small lipophilic molecules so as to isolate and monitor their function individually.

The surface optical technique surface plasmon resonance spectroscopy (SPS) can be employed to monitor interaction processes at the tethered artificial membrane if the solid substrate is a metal layer. SPS has become interesting for biosciences in the context of biorecognition and binding studies at functional surfaces in contact to aqueous solutions containing biomolecules [3], [4], [5]. There are several commercial instruments available that exploit surface plasmon resonance (SPR) for detection of (bio)molecular interactions [3], [6].

By combining SPS with surface plasmon enhanced fluorescence spectroscopy (SPFS), we gain sensitivity when detecting binding events at tethered membranes. Depending on the size and shape of the fluorescent molecule that is detected, an enhancement factor of up to 1000 can be achieved in comparison to SPS [7], [8]. This opens up a new dimension of sensitivity in observing interaction processes at interfaces.

In this review, we describe the instrumental setup for simultaneous SPS and SPFS, the design of suitable flow cuvettes and different modes of data acquisition. We focus on the in situ setup of tethered artificial membranes that are specifically biofunctionalized for our investigations.

Section snippets

Surface plasmon resonance (SPR)

The theoretical background of SPR is described in detail in the works of Knoll [9] and Raether [10]. Surface plasmon resonance occurs at the boundary of two materials of different optical properties described by their different dielectric functions, e.g., at the interface of a thin noble metal film with a negative dielectric function and a dielectric medium with a positive dielectric function (Fig. 1A) [9], [10]. If p-polarized incident light strikes the metal film at the resonance angle (θ_SPR

Application of the method

The classical configuration of an SPS/SPFS binding assay with tethered artificial membranes involves a membrane protein immobilized in the artificial lipid bilayer and a free fluorescently labeled ligand. The functional incorporation of the integral membrane protein in the lipid bilayer is monitored in real-time by SPS. The interaction of this membrane protein with a soluble, fluorescently labeled ligand from the aqueous phase is observed by SPFS. Classical SPS/SPFS binding assays were used to

Concluding remarks

As was outlined in this review, the combination of SPS/SPFS with tethered artificial membranes provides a powerful tool to analyze membrane-related processes such as ligand binding by integral membrane proteins or interaction of soluble proteins with membrane lipids. Despite the high sensitivity of SPS/SPFS for monitoring, on-line and in real-time, small changes in the thickness of surface coatings, e.g., by adsorption/desorption processes, it is a relatively simple technique. The fact that

Acknowledgments

We thank Danica Christensen and Nediljko Budisa for critically reading the manuscript. Ulrich Männl is acknowledged for technical assistance with the flow cuvette. Special thanks are extended to Andreas Scheller for excellent software support and maintenance.

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Binding assays with artificial tethered membranes using surface plasmon resonance

Abstract

Introduction

Section snippets

Surface plasmon resonance (SPR)

Application of the method

Concluding remarks

Acknowledgments

Curr. Opin. Biotech.

J. Biotechnol.

Colloid. Surface. A.

Curr. Opin. Biotech.

Colloid. Surface. A.

Anal. Chim. Acta.

Curr. Opin. Biotech.

Anal. Biochem.

Biosens. Bioelectron.

Biophys. J.

Biophys. J.

Anal. Chem.

Biosens. Bioelectron.

Biosens. Bioelectron.

Cell

Chem. Biol.

J. Mol. Recognit.