Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

1-POS Board 1 FRET-BASED ANALYSIS OF CONFORMATIONAL CHANGES IN SYNTAXIN-1 ON THE PLASMA MEMBRANE Kazuki Obashi 1 ; Justin W Taraska 1 ; 1 National Heart, Lung, and Blood Institute, Bethesda, MD, USA Calcium-triggered exocytosis of neurotransmitters and hormones from neurons and neuroendocrine cells is regulated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Syntaxin-1 is the plasma membrane t-SNARE. It has been proposed to adopt two conformations: open and closed. In the closed form, the Habc domain binds to the SNARE motif and prevents the formation of the SNARE complex. To assemble into the fusion-competent SNARE complex, syntaxin-1 is thought to adopt the open form. Thus, conformational changes in syntaxin-1 are key to the regulation of exocytosis and understanding the structural dynamics and molecular interactions of this protein inside cells is necessary to construct a mechanistic model of exocytosis. Yet, the conformational dynamics of syntaxin-1 in relation to vesicle docking, SNARE complex formation, and fusion at the plasma membrane are not fully understood. Here, we develop and use a short-range FRET method that employs site-specifically labeled with un-natural fluorescent amino acids donor fluorophores and transition metal ion acceptors engineered into sites within the proteins. We further use unroofed membranes from PC12 cells to allow biochemical access to the SNARE complexes on the intracellular side of the plasma membrane. Here, we report our progress in developing the imaging methods to analyze the conformational dynamics of syntaxin-1. We discuss how the conformations of syntaxin-1 are changed by molecular interactions and the induction of exocytosis.

53