Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Abstract

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods.SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein). Video LinkThe video component of this article can be found at

The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory and improved activity

“…Like all ABC importers BtuC 2 D 2 must associate with its cognate SBP since it is the latter that binds the substrate and delivers it to the transporter. We measured the interaction between BtuC 2 D 2 that had worked or rested at 28 °C and BtuF using Surface Plasmon Resonance 29 . We loaded identical amounts (by mass) of worked or rested BtuC 2 D 2 onto a biosensor chip and then washed the chip 50,000-fold to remove any residual ATP.…”

“…The procedure for measuring the interaction between BtuC 2 D 2 and BtuF using SPR had been extensively described 28 29 . Briefly, BtuC 2 D 2 (15–20 ng, 1500–2000 RU) was immobilized onto a Ni-NTA biosensor chip (GE healthcare) and FLAG-tagged BtuF was injected at the indicated concentration.…”

ATP binding and hydrolysis disrupt the high-affinity interaction between the heme ABC transporter HmuUV and its cognate substrate-binding protein

“…All measurements were performed using a Biacore T200 (GE Healthcare) as extensively as described before (34,35,60). His-HmuUV, His-BtuCD, and His-MolBC were immobilized directly onto nickel-nitrilotriacetic acid biosensor chip (GE Healthcare).…”

Surface Plasmon Resonance technology to assess biological interactions

“…The sensor chip typically used in BIAcore system, that employs the Kretschmann con iguration [16,17], is a dextran matrix, composed of a thioalkane linker layer (~100 nm thick), that creates an hydrophilic environment for different chemical methods of ligand immobilization, which usually require an amount of ligand immobilized (about 10,000 RU) that corresponds to approximately ≈70 fmol/mm2, and for binding process [18]. The activation of the gold layer and the application of a speci ic coat avoid non-speci ic binding of biomolecules to the activated surface [19]. Furthermore, the dextran matrix allows both ligand immobilization with minimal conformational distortion and maximum access of the binding epitopes of ligand and analyte.…”