SNARE (Soluble NSF (N-ethylmaleimide Sensitive Fusion) Attachment Protein Receptors) proteins
have been linked to the membrane fusion mechanism since 1993 as fusion proteins and have been
suggested to be the minimal machinery. The complexity of the fusion process means that many
questions remain unanswered as to how SNARE proteins perform their role. The most favoured
model (the stalk model) does not involve proteins directly and so the influence of the SNARE
proteins on lipid properties is of interest. In this thesis, work is presented which investigates how
these proteins may manipulate membrane properties in order to promote fusion.
Purified proteins solutions of His6-VAMP2 (Vesicle Associated Membrane Protein 2), His6-SNAP-25
(Synaptosomal-associated Protein 25) and a truncated form of Syntaxin 1A (His6-ΔN-Syx 1A, w.t aa
181-288) were obtained following bacterial over-expression. Fluorescent versions of His6-VAMP2
and His6-ΔN-Syx 1A were produced by the addition of cysteine residues to the C-terminus followed
by labelling using Alexa Fluor® 488-C5-maleimide and Alexa Fluor® 555-C2-maleimide respectively.
These fluorescent proteins were used to establish that the purified protein inserted into model lipid
bilayers.
The effect of SNARE protein incorporation on the relaxed curvature of bilayers was explored by
examining giant unilamellar vesicles grown using electroformation. Bilayers containing either 1:300
His6-VAMP2: DOPC or 1:1:600 His6-SNAP-25: His6-ΔN-Syx 1A: DOPC were smaller than pure DOPC
vesicles, indicating that SNARE proteins increase the relaxed curvature of the bilayer. Analysis of
these vesicles by micropipette aspiration suggested that VAMP2 lowered the bending rigidity of the
membrane and a reduction in the area expansion modulus relative to the pure lipid bilayer was
found. The t-SNARE sample also indicated a reduction in bending rigidity but the area expansion
modulus was found to increase. These latter results are thought to be due to the formation of
protein aggregates.
Lipid mixing assays were conducted to investigate how changes in the properties of liposome
bilayers affected fusion rates. It was found that the addition of DOPE to DOPC bilayers increased the
rate of hemifusion and this was also found for cholesterol addition, suggesting both components are
fusogens. The rate of hemifusion rose continually upon DOPE addition but reached a plateau in the
cholesterol study shortly after 10 mol%. Despite this, the fusion rates for the cholesterol study were
generally higher than the same mol% DOPE added. The changes in fusion rates observed have been
explained by considering the impact of the additives on the free energy and stored curvature elastic
stress of membranes as well as the change in the energy of formation of intermediate structures. From the findings of this thesis it is proposed that the SNARE proteins are able to soften the
membranes in which they reside. This allows the membrane to be deformed with less energy input.
The strength of the SNARE complex and the force applied to the membranes during its formation
increases membrane tension and reduces inter-membrane separation; promoting hemfusion.
Following the action potential of the neuron it is proposed that a conformational change occurs in
the synaptic SNARE complex, pulling on the hemifusion diaphragm and inducing the formation of a
fusion pore.