(184p) Energetics of SNARE Dissociation

Abstract

SNAREs are tail-anchored membrane proteins which play an integral role in membrane fusion [1]. The SNARE family is composed of two target membrane bound (t)-SNARE proteins (syntaxin-1A and SNAP-25) and one synaptic vesicle bound (v)-SNARE protein (VAMP-2). Bringing the target membrane and synaptic vesicle into close contact with each other is an energetically unfavorable process as the two bilayers are very stable and carry an overall negative charge [2]. SNARE proteins are known to cross over this energetic barrier by utilizing the energy generated during formation of a highly thermostable ternary SNARE complex [1, 3]. The complex is a coiled-coil bundle of four alpha-helices, two of which are contributed by SNAP-25 and one each by syntaxin-1A and VAMP-2 [4-6]. The amount of energy stored in this complex is an important parameter to understand the overall thermodynamics of the fusion process and can be related to the energy required for dissociation of SNARE complex. Dissociation experiments have been performed in vitro [7, 8] using AFM (Atomic Force Microscopy) and SFA (Surface Force Apparatus) and provide a measure of free energy associated with this process. In recent years, Steered Molecular Dynamics (SMD) technique has been applied to study protein unfolding and dissociation [9-11] by applying pulling force at one terminal while keeping the other terminal fixed. SMD simulations mimic AFM-based experiments but proceed on much smaller time scales [12]

To provide molecular-level insights on the SNARE dissociation process, SMD simulations are performed in this work by pulling VAMP-2 protein out of the SNARE complex thereby dissociating it from syntaxin-1A and SNAP-25. Pulling force is applied at N-terminal of VAMP-2 while the C-terminal of (t)-SNAREs are fixed. Pulling is performed at a constant velocity of 12.0 Å/ns with the force being evaluated at every 2 ps. Distinctive peaks are observed in the force curve marking specific dissociation events which can be associated with breakage of intra/inter-molecular hydrogen bonds or hydrophobic interactions. The dissociation event is visualized with VMD [13] software and atomic-level analysis is performed at every force-curve peak point to investigate the type of bond-dissociation and atoms / amino-acids involved. To understand if the dissociation pathway and dynamics is rate-dependent, the experiment is also performed with a pulling velocity of 120.0 Å/ns and the force curves are compared. The Potential of Mean force (PMF) is evaluated for the dissociation process providing a bridge between the non-equilibrium nature of SMD method and the real-life equilibrium experiments.

References

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