Free Energy Landscapes of Vesicle Fusion by Umbrella Sampling MD Simulations

Abstract

Despite intensive investigation, the energy landscapes governing membrane fusion in vitro and in vivo remain uncertain. A plethora of factors including small molecules, ions, fusion proteins, and osmotic pressure gradients are known to influence fusion rates, but these perturbations only hint at the underlying molecular mechanisms.The barriers and metastable structures that characterize fusion free energy landscapes are inherently difficult to resolve atomistically due to the fluid, disordered nature of membranes. These pathways are also difficult to access with molecular resolution simulations, namely molecular dynamics (MD), due to the time scales associated with spontaneous fusion and the lack order parameters capable of driving fusion progress through high energy intermediates.To address this challenge, we have developed a novel umbrella sampling method paired with an order parameter capable of driving and controlling fusion progress. Our initial results for 20 nm POPC vesicles give a barrier of 43 kBT along a pathway beginning as a metastable stalk, proceeding over a barrier with a hemifused structure and then ending as an opened fusion pore. Though marginally metastable, the hemifusion diaphragm does not expand, likely due to the small vesicle size and the lack of a lipid reservoir, but instead either reverts back to a stalk or proceeds forward to form a fusion pore.