Redox of Cysteines and Protein Folding of Snap-25

The SNARE proteins SNAP-25, syntaxin, and VAMP play critical roles in neuronal exocytosis by providing the four helical regions (SNARE domains) that form a coiled-coil complex required for neurotransmitter release. This complex is continually formed and unwound as exocytotic vesicles fuse and are recycled. Using multiple techniques, we observe clear structural changes in SNAP-25B due to temperature, pH, ionic strength, methanol, and oxidation state. Here we focus on redox state and structural changes due to temperature and methanol. Using Circular Dichroism to measure the secondary structure of SNAP-25B in low ionic strength solutions, we observe an increase in alpha-helical structure when methanol is added, or the temperature is lowered, similar to the helical shift observed when SNAP-25 forms a complex with syntaxin and VAMP. Methanol (30%) or heating (34C) also breaks up large aggregates of SNAP-25B, as observed by decreased opacity in the CD signal and a loss of high molecular weight bands in non-denaturing gels. The four cysteines in SNAP-25B are thought to be palmitoylated in vivo and can be modified by addition of iodoacetic acid (IAA). Using non-denaturing gels, we observe a shift of mobility of SNAP-25B depending on the redox potential of the reaction solution. We previously showed that these cysteines are oxidized by micromolar Cu 2+ by measuring the number of cysteines accessible for labeling with a Biotin-maleimide tag technique developed in our lab (Woodbury et al., 2011. Anal. Biochem. 417:165-173). These data support the hypothesis that SNAP-25B structure and cysteine accessibility are sensitive to environmental changes, which may modulate SNAP-25 palmitoylation and neurosecretion in vivo