Integrative Structural Dynamics Probing of the Conformational Heterogeneity in Synaptosomal-Associated Protein 25

Abstract

SNAP-25 (synaptosomal-associated protein of 25 kDa) is a prototypical intrinsically disordered protein (IDP) that is unstructured in its unbound form but forms coiled-coil helices in the SNARE complex. With high conformational heterogeneity, detailed structural dynamics of unbound SNAP-25 remains elusive. Here, we present an integrative method to probe the structural dynamics of SNAP-25 by combining replica-exchange discrete molecular dynamics (rxDMD) simulations and label-based experiments at ensemble and single-molecule levels. The rxDMD simulation allows us to systematically characterize the coil-to-molten-globular transition and reconstruct structural ensemble consistent with prior ensemble experiments. Label-based experiments using Förster Resonance Energy Transfer and Double Electron Electron Resonance further probe the conformational dynamics of SNAP-25. Agreements between simulations and experiments under both ensemble and single-molecule conditions allow us to assign specific helix-coil transitions in SNAP-25 that occur in sub-millisecond timescales and potentially play a vital role in forming the SNARE complex. We expect this integrative approach would help further our understanding of IDPs.

Keywords: Intrinsically disordered proteins, conformational switching, single-molecule FRET, EPR, DEER, replica-exchange discrete molecular dynamics