Use of Surface Plasmon Resonance (SPR) to Determine Binding Affinities and Kinetic Parameters Between Components Important in Fusion Machinery.

ABSTRACT

Surface plasmon resonance (SPR) can be used to analyze both binding affinities and kinetic parameters between a ligand and an analyte. SPR can be performed by either cross-linking a given ligand to a sensor chip covalently or utilizing high-affinity non-covalent interactions to secure a ligand in a particular conformation to a chip, both of which have their potential advantages. SPR measurements are mass based and reflect the proportional amount of analyte bound to a given ligand at a given concentration when flowed at a set rate in order to determine the binding parameters of a given biochemical interaction. The resultant sensorgram can indicate different types of binding events as well as provide both ka and kd, which can be used to determine an equilibrium dissociation constant KD. SPR can be used to measure binding affinity of proteins involved in fusion such as between SNAREs, SNAREs, and proteins that interact with them such as Sec18 (NSF) or Sec17 (alpha-SNAP), or to measure the binding of any fusion-related protein to a specific lipid or other small molecules; however, KDs are determined by SPR using a titration of concentrations of analyte and a maximum point on the sensorgram signifying saturation of the protein in order to determine a steady-state KD.