Abstract
SNAP-25, together with other SNARE proteins, drives fusion of synaptic vesicles with the nerve cell membrane, leading to neurotransmitter release. It is unique in contributing two α helices to the four-helix bundle known as the SNARE complex. Complex formation drives fusion as these proteins transform from a disordered to ordered (coiled-coil) state. SNAP-25 has two isoforms, -25A and -25B, but little is known of any structural differences, nor are there extensive reports of the structures of its two helical domains, SN1 and SN2. Thus, the benefit of having two distinct isoforms of SNAP-25, each with two distinct domains, is unknown. Here, we use circular dichroism spectroscopy and mass spectrometry to further characterize the secondary structure of SNAP-25A, SNAP-25B, SN1, SN2, and a cysteine-free version of SNAP-25A. We demonstrate that these proteins undergo structural transitions, with changing fractions of α helix, β sheet, and random coil. These different structures can be induced by varying the environmental conditions of ionic strength, pH, temperature, or redox state. We use triangle plots to directly display the change in ternary composition following changes in these four parameters. We report that SNAP-25A and SNAP-25B make distinctly different structural changes. We show that the secondary structure of SN1 is more variable than SN2. These data add to the ongoing literature characterizing SNAP-25 as an intrinsically disordered protein that is sensitive to environmental conditions in neuronal cells and may function as a redox sensor to modulate neurotransmitter release.