Use of Bio-Layer Interferometry (BLI) to Measure Binding Affinities of SNAREs and Phosphoinositides

Abstract

Bio-Layer Interferometry (BLI) is a technique that uses optical biosensing to analyze interactions between molecules. The analysis of molecular interactions is measured in real-time and does not require fluorescent tags. BLI uses disposable biosensors that come in a variety of formats to bind different ligands including biotin, hexahistidine, GST, and the Fc portion of antibodies. Unlike surface plasmon resonance (SPR), BLI is an open system that does not require microfluidics, which eliminates issues that result from clogging and changes in viscosity. Importantly, BLI readings can be completed in minutes and can be formatted for high throughput screening. Here we use biotinylated short chain phosphoinositides and phosphatidic acid bound to streptavidin BLI biosensors to measure the binding of the soluble Qc SNARE Vam7 from Saccharomyces cerevisiae. Unlike most SNAREs, Vam7 lacks a transmembrane domain or lipid anchor to associate with membranes. Instead Vam7 associates to yeast vacuolar membranes using its N-terminal PX domain that binds to phosphatidylinositol 3-phosphate (PI3P) and phosphatidic acid (PA). Using full length Vam7, Vam7^Y42A, and PX domain alone, we determined and compared the dissociation constants (K_D) of each to biotinylated PI3P and PA biosensors.

Keywords: PX; Phosphatidic acid; Phosphatidylinositol; SNARE; Vam7.