Protein-Protein Interactions: Surface Plasmon Resonance.

Abstract

Surface plasmon resonance (SPR) is one of the most commonly used techniques to study protein-protein interactions. The main advantage of SPR is it gives on the ability to measure the binding affinities and association/dissociation kinetics of complexes in real time, in a label-free environment, and using relatively small quantities of materials. The method is based on the immobilization of one of the binding partners, called the ligand, on a dedicated sensor surface. Immobilization is followed by the injection of the other partner, called the analyte, over the surface containing the ligand. The binding is monitored by subsequent changes in the refractive index of the medium close to the sensor surface upon injection of the analyte. During the last 10 years, SPR has been intensively used in the study of secretion systems because of its ability to detect highly dynamic complexes that are difficult to investigate using other techniques. This chapter will guide users in the setup of SPR experiments in order to identify protein complexes and to assess their binding affinity or kinetics. It will include detailed protocols for (i) the immobilization of proteins with the amine coupling capture method, (ii) analyte-binding analysis, (iii) affinity/kinetic measurements, and (iv) data analysis.Secretion systems are multiprotein complexes allowing the transport of a large number of effectors from the inside to the outside of bacterial cells. The assembly of these supramolecular machineries is ensured by the formation of protein complexes with extremely different times of stability, from transitory to stable interactions. To understand the function of these machineries as well as their modes of association, it is important to study their building blocks by identifying the different interacting partners and assessing their relative affinities and association/dissociation kinetics. For that purpose, scientists combine genetic, biochemical, and biophysical tools. During the last decade, the use of surface plasmon resonance (SPR) in the study of secretion systems has increased spectacularly [1-12]. This in vitro approach is the method of choice to study such dynamic systems owing to its ability to detect both weak and strong interactions ranging from the millimolar to the nanomolar range [13, 14]. SPR can be used as a primary tool to screen interacting partners or as a validation tool for interactions previously identified by other methods (e.g., bacterial two-hybrid, co-immunoprecipitation, chemical crosslinking). The determination of the affinity or kinetics of an interaction, as can be done by SPR, is fundamental to understanding the nature of binding at the cellular level.