Structural Changes in the SNARE Protein SNAP&25 by PH and Ionic Strength

Abstract

The SNARE protein SNAP-25 plays a critical role in neuronal exocytosis by providing 2 of the 4 helical regions (SNARE domains) that form a coiled-coil quaternary structure. SNAP-25B contains four cysteines, one tryptophan, and one tyrosine all found in the linker region. Using Circular Dichroism to measure the secondary structure of SNAP-25B, we observe significant structural changes with pH and ionic strength. Three fundamental structures are observed: alpha helix, random coil, and beta sheet. The alpha helix and random coil have been observed previously, but the beta sheet (which is red-shifted to ∼229nm) is observed at pH5.5 and >75mM salt (NaF). The beta-sheet is reversibly changed into alpha helix at pH9. Using the fluorescent signal from tryptophan and tyrosine, an increase in pH reversibly changes the peak emission with a pK of ∼8.5, similar to the expected pKa for the sulfhydryl of cysteine. Replacing all cysteines with serines prevents this shift. The conformational changes observed above may lead to a change in accessibility of the 4 cysteines in SNAP-25B linker. Consistent with this hypothesis, we observe that decreasing ionic strength varies 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 the structure of SNAP-25B is sensitive to ionic strength and pH, which may contribute to modulation of exocytosis in vivo.