Biophysical Studies of SNARE Protein- Membrane Interactions

Abstract

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.