Search

Vacuole fusion is driven by SNARE proteins that require activation-or priming-by the AAA+ protein Sec18 (NSF) before they can bring membranes together and trigger the merger of two bilayers into a continuous membrane. Sec18 resides on vacuoles prior to engaging inactive cis-SNARE complexes through its interaction with the regulatory lipid phosphatidic acid (PA). Binding PA causes Sec18 to undergo large conformational changes that keeps it bound to the membrane, thus precluding its interactions with SNAREs. Such conformational changes can be measured by various biochemical and biophysical assays. The conversion of PA to diacylglycerol by the PA phosphatase Pah1 releases Sec18 from the membrane-bound pool and promotes its transfer to SNARE complexes allowing priming to occur. Here we describe four spectroscopy-based methods to distinguish conformational changes from alterations in secondary structure during PA binding. These methods only require purified protein and short chain soluble lipids, making the methods rapid and affordable ways to screen the effects of specific protein-lipid interactions. The assays described in this chapter include 1-anilino-8-naphthalenesulfonate (ANS) spectroscopy and intrinsic tryptophan fluorescence to detect exposure of hydrophobic regions; differential scanning fluorometry to measure changes in protein stability across a temperature gradient; and circular dichroism to examine changes in secondary structure., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)